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Destruction of acid- and non-adapted Listeria monocytogenes during drying and storage of beef jerky
Food Microbiology, 2002, 19, 545^559 Available online at http://www.idealibrary.com on
doi:10.1006/yfmic.510
ORIGINAL ARTICLE
Destruction of acid- and non-adapted Listeria monocytogenes during drying and storage of beef jerky Mehmet Calicioglu1, John N. Sofos 1; *, John Samelis1, Patricia A. Kendall2 and Gary C. Smith1
Presence of Listeria monocytogenes in ready-to-eat meat products is not desired and strictly regulated in the US. Inactivation of acid- and non-adapted L. monocytogenes inoculated on beef slices was studied during drying and storage of jerky formulated with modi¢ed marinades. The inoculated (¢ve-strain composite, c. 6?2 log cfu cm 2) slices were subjected to marinades (41C, 24 h) prior to drying (601C for 10 h) and aerobic storage (251C for 60 days). The predrying marinade treatments tested were, ¢rst, no treatment, control (C); second, traditional marinade (TM); third, double amount of TM modi¢ed with 1?2% sodium lactate, 9% acetic acid, and 68% soy sauce containing 5% ethanol (MM); fourth, dipping into 5% acetic acid for 10 min and then applying theTM (AATM); and ¢fth dipping into 1% Tween 20 for 15 min and then into 5% acetic acid for 10 min followed by theTM (TWTM). Bacterial survivors on beef slices were determined during drying and storage using tryptic soy agar with 0?1% pyruvate (TSAP), and PALCAM agar. Results indicated that drying reduced bacterial populations in the order of pre-drying treatments TWTM (5?9^6?3 log cfu cm 2 in 10 h)ZAATMZMM4TMZC (3?8 4?6 log cfu cm 2 in 10 h). No signi¢cant (PZ0?05) di¡erence was found in inactivation of acidadapted and non-adapted inocula within individual treatments. Bacterial populations dropped below the detection limit ( 0?4 log cfu cm 2) as early as 4 h during drying or remained detectable even after 60 days of storage depending on acid-adaptation, predrying treatment, and agar media. These results indicated that acid-adaptation may not increase resistance to microbial hurdles involved in jerky processing and that use of modi¢ed marinades may improve the e¡ectiveness of drying in inactivating L. monocytogenes. # 2002 Elsevier Science Ltd. All rights reserved. Received: 28 February 2002
Introduction Dried products, such as jerky, have been considered as one of the safest food groups for humans because their manufacture involves hurdles to microbial survival or growth. Such
*Corresponding author. Fax: +1 970 491 0278. E-mail: [email protected] 0740 -0020/02/060545+15 $35.00/0
hurdles include low water activity (o0?85), drying temperature, and use of preservatives such as salt, organic acid and sodium nitrite, depending on the composition of the marination mixture (Gailani and Fung 1986). However, a recent report (Levine et al. 2001) by the Food Safety and Inspection Service of the United States Department of Agriculture (FSIS/ USDA) indicated that for the period 1990^ 1999, cumulative prevalence of Salmonella and r 2002 Elsevier Science Ltd. All rights reserved.
1 Department of Animal Sciences, Center for Red Meat Safety, Colorado State University, Fort Collins, Colorado 80523, USA 2 Department of Food Science and Nutrition, Colorado State University, Fort Collins, Colorado 80523, USA
546 M. Calicioglu et al.
Listeria monocytogenes in jerky produced in federally inspected plants was 0?31% and 0?52%, respectively. Currently, FSIS applies a‘zero-tolerance’ policy for L. monocytogenes in ready-toeat meat products including jerky. Products that are not in compliance with this policy are considered to being adulterated under the provisions of United States Federal Meat Inspection Act (21 US Code, 601 (m)). The recent Salmonella and Escherichia coli O157:H7 outbreaks linked to jerky (CDC 1995, Keene et al. 1997) have renewed interest in evaluating the e⁄cacy of jerky processing, especially when prepared in home-type dehydrators, for inactivating pathogens (Albright 2000, Harrison and Harrison 1996, Harrison et al. 2001, Keene et al. 1997). As a response to the problem, the USDA/FSIS (1998) has recommended cooking meat to 71?11C before drying to eliminate the risk of pathogen survival. However, traditional jerky drying (601C, 10 h) after marination, irrespective of predrying heating, has been shown to be e¡ective in reducing (e.g., 5 -log unit reduction) E. coli O157:H7, Salmonella typhimurium and L. monocytogenes (Harrison and Harrison 1996; Harrison et al. 2001). In contrast, Keene et al. (1997) reported that drying at r63o C was not a reliable method for eliminating E. coli O157:H7 in marinated (pH 4?2) whole muscle venison jerky. Preheating of meat and/or drying of jerky at high temperatures for extended periods of time may result in a product that di¡ers texturally from traditional jerky and, thus, it may have reduced consumer acceptability. There is a need for development of new food preservation strategies with the objective of inactivating bacteria while avoiding severe treatments that change desired characteristics of the food, because most traditional food preservation methods (e.g. chilling, freezing, drying) slow down or inhibit growth of bacteria rather than inactivating them (Gould 2001). Leistner (2000) indicated that preservation methods/ technologies that simultaneously or sequentially expose bacteria to multiple stress factors (hurdles) lead bacteria to metabolic exhaustion or homeostatic disturbance followed by cellular death. Thus, employing intervention strategies that sensitize bacteria attached to meat slices via predrying treatments to drying at
mild temperatures (e.g. 60o C) can be a viable option to avoid severe heat treatments. Examples of such interventions might be weakening bacterial attachment by use of surfactants followed by dipping in organic acid solution prior to marination and drying or designing new marinade solutions that can disturb or damage bacterial homeostasis. These marinades may provide residual antimicrobial e¡ects during storage against possible post-processing contamination. There is some evidence that acid-adaptation of L. monocytogenes may enhance its survival in acidic foods as well as increase cross-protection to other type of stresses (Buchanan et al. 1994, Gahan et al. 1996, Lou and Yousef 1997, Saklani-Jusforgues et al. 2000, Francis and O’Beirne 2001).To date, it is not known whether acid-adapted pathogenic bacterial cells survive better than non-acid-adapted cells during the jerky-making process. Therefore, the objective of the present study was to evaluate the e¡ectiveness of various chemical-based predrying treatments (modi¢ed marinades) in enhancing destruction of acid- or non-adapted L. monocytogenes cells during drying and storage of whole muscle beef jerky.
Materials and Methods Bacterial strains used and preparation of inoculum A ¢ve-strain composite of L. monocytogenes was used for inoculating beef strips. These strains were LM101 (sausage isolate, serotype 4b), LM103 (sausage isolate, serotype 1a), N7143 (meat isolate, serotype 3a), N-7144 (meat isolate, serotype 1/2a), and TB2000 (turkey breast isolate, unknown serotype). Each strain was propagated (301C, 24 h) and maintained on tryptic soy agar (TSA, Difco Laboratories, Detroit, Michigan, USA) slants at 41C. Strains were subcultured monthly. The cultures were activated by transferring a loopful of each strain into 9 ml of tryptic soy broth (TSB, Difco) and incubating at 301C for 24 h. Acid-adaptation was induced by preculturing strains in media with glucose, a condition previously reported to result in increased acid tolerance as
Jerky processing to inactivate Listeria 547
compared to L. monocytogenes cultures grown without glucose (Buchanan et al. 1994). More speci¢cally, a 0?1-ml portion of activated culture of each strain was transferred into 9 -ml tubes of glucose-free TSB (TSB-G) (BBL, Becton Dickinson Co., Cockeysville, Maryland, USA) for non-acid-adapted cells (Buchanan et al. 1994) and in glucose-free TSB with 1% glucose (Sigma, St. Louis, Missouri, USA) added (TSB+G) for acid-adapted cells (Samelis et al. 2001c). Addition of 1% glucose was aimed at enhancing acid-adaptation of TSB+G cultures, although 0?3% glucose added to brain^heart infusion (BHI) broth, or even 0?2% or 0?25% glucose as part of the normal formulation of BHI and TSB, respectively, have been found su⁄cient to deliver signi¢cant increases in acid tolerance compared to TSB-G cultures of L. monocytogenes (Buchanan et al. 1994, Samelis et al. 2001a). The challenge method used to assess acid tolerance was described previously (Samelis et al. 2001a). Con¢rmatory experiments during previous studies (Samelis et al. 2001c) have shown that with 1% glucose added to TSB-G, the pH values of the resulting acid-adapted cultures (301C, 24 h) of L. monocytogenes strains N-7144 and N7143 were 4?4^4?5 as compared with pH values of 6?6^6?7 of their non-adapted counterparts grown in TSB-G. This induced acid-adaptation via glucose fermentation by L. monocytogenes provided signi¢cant protection to the pathogen following a 2 -h exposure to acidi¢ed TSB with lactic acid, pH 3?5 (e.g. 1?0^1?3 log population reductions) as compared to the corresponding non-adapted populations (Z3?9 -log reductions) (unpublished data). Thus, the TSB+G and TSB-G cultures of L. monocytogenes strains used in this study were acidand non-adapted, respectively. After incubation at 301C for 22^24 h, individual cultures were combined in a sterile tube for centrifuging at 6000 rpm (2900 g) (Eppendorf, model 5402) for 30 min at 41C. The resulting pellet was washed once with 0?1% phosphatebu¡ered saline (PBS, Sigma) to remove residual organic material, recentrifuged, and then resuspended in PBS to a ¢nal volume of 100 ml. The average cell concentration in the resulting composite inoculum was 7?0 log cfu ml 1 .
Preparation of meat slices Vacuum-packaged and frozen (^181C) beef inside rounds (o3 months) were purchased from the Colorado State University Meat Science Laboratory (Fort Collins, Colorado, USA). Following thawing at 41C overnight, inside rounds were sliced at 0?6 -cm thickness using a food slicer (model 610, Hobart Corp., Troy, Ohio, USA) and cut into pieces of 8?7 4?0 cm2 using a plastic template and knife. Approximately, 100 slices (2?2 kg) of meat were vacuum-packaged and kept frozen at ^181C until use (1 ^ 3 weeks).
Inoculation procedure Frozen beef slices were thawed at 41C for 24 h, placed on plastic trays and inoculated under a laminar-£ow hood. Portions of 0?5 ml of the L. monocytogenes inoculum were placed on the upper surface of each slice and spread onto the entire surface area using a sterile bent glass rod. Bacteria were allowed to attach to the meat surface for 15 min at ambient temperature. The slices were then £ipped over and the other side was inoculated following the same procedure. The resulting level of inoculum was approximately 6?2 log cfu cm 2 .
Predrying treatments They included ¢rst, control, no treatment (C); second, marination with traditional marinade (pH 4?3) (TM); third, increased (double the amount) marination with modi¢ed marinade (pH 3?0) (MM), fourth, dipping into 5% acetic acid solution followed by traditional marination (AATM); and ¢fth, sequential 15 -min immersion in 1% Tween 20 solution (polyoxyethylene-20 -sorbitan monolaurate) (pH 6?6), then 10 -min immersion in 5% acetic acid solution (pH 2?5) followed by marination with TM (TWTM). The TM was prepared for 1?0 kg of meat (Andress and Harrison 1999) as follows: 60 ml soy sauce (Kikkoman Foods, Walworth, Wisconsin, USA), 15 ml Worcestershire sauce (Heinz, Pittsburgh, Pennsylvania,USA), 0?6 g black pepper (Heller Seasoning and Ingredients Inc., Chicago, Illinois, USA), 1?25 g garlic powder (Excalibur Seasoning Co. Ltd. Pekin, Illinois, USA), 1?5 g onion powder (Excalibur),
548 M. Calicioglu et al.
4?35 g old hickory smoked salt (Tone Brothers Inc., Ankeny, Iowa, USA). In the present study, 30 ml of this marinade was used for 450 g of inoculated meat. Marinade was spread manually on beef slices and then the slices were manually mixed to cover the entire surface area using £ame-sterilized forceps. The modi¢ed marinade was prepared for 1?0 kg of meat as follows: 120 ml of ‘‘milder soy sauce’’ (Kikkoman) containing approximately 4?7^5?0% ethanol as preservative, 30 ml of Worcestershire sauce, 0?6 g black pepper, 1?25 g garlic powder, 1?5 g onion powder, 4?35 g smoke-£avored salt, 3?6 ml of 60% food-grade sodium-L -lactate preparation (Purac Inc., Lincolnshire, Illinois, USA), and 16 ml of glacial acetic acid (Mallinckrodt Baker Inc., Paris, Kentucky) to adjust to pH 3?0. A 60 -ml portion of this marinade solution (double that of TM) was spread on 450 g of beef, and mixed to cover surfaces of meat slices. For the fourth treatment (AATM), meat slices were dipped into 5% (v/v) acetic acid solution prepared using glacial acetic acid at ambient temperature for 10 min (450 ml per 450 g of meat). These slices were drained for 2 min to remove excessive £uid using an empty dehydrator tray and placed on a tray covered with aluminum foil followed by marination with TM. For the ¢fth treatment (TWTM), meat slices were dipped into 1% (v/v) Tween 20 (Fisher Scienti¢c Inc., Fair Lawn, New Jersey, USA) solution (450 ml per 450 g of meat) for 15 min at ambient temperature in a 1000 ml glass container. These slices were drained for 2 min, and then the same steps as were used for treatment AATM were followed. Black pepper, garlic and onion powder were irradiated by their manufacturers. Following each treatment, trays containing the inoculated and marinated slices were covered with aluminum foil and held at 41C for 24 h prior to drying.
Drying After the 24 h of refrigeration, the inoculated and treated meat slices were dried at 601C for 10 h in a home-type dehydrator (American Harvest Gardenmaster, model FD-1000, Nesco, Chaska, Minnesota, USA; two appliances used). The dehydrators were cylindrical in shape and were composed of a base unit and
three drying trays. The dehydrator base unit generated hot air, which ventilated upward through the sides and a hole in the middle of the trays. The target temperature was based on the air temperature measurement taken from the middle hole of the dehydrator. The dehydrators with empty trays were preheated for approximately 20 min to 601C (1401F). The empty trays were then replaced with other trays preloaded with meat slices. During drying, the temperature of the dehydrator through the middle hole and the surface temperature of meat slices on each of the bottom, middle, and top trays were monitored using thermocouples (Type K beaded probes, Pico Technology Ltd. Cambridge, UK) and real-time data recording software (Pico Technology). After drying, the jerky strips were held in the dehydrators overnight as recommended by Andress and Harrison (1999), and then placed into 24 -oz WhirlPak sterile plastic bags (Nasco, Fort Atkinson, Wisconsin, USA) for storage at ambient temperature (25711C).
Analysis Two samples (1 slice sample 1 ) per treatment were aseptically transferred into sterile-plastic bags (Nasco, Modesto, California, USA) at each sampling interval. These intervals included after inoculation, and 0 (after marination and 24 h refrigeration), 4, 7 and 10 h during drying for each treatment, and days 15, 30 and 60 during storage. A 25 ml portion of 0?1% sterile bu¡ered peptone water (BPW) (Difco) was added to each sample bag prior to pummeling for 2 min at ambient temperature. Serial decimal dilutions were made using 9 -ml BPW tubes and 0?1-ml portions were surface plated onto each of duplicate plates of each agar medium. Bacteria were enumerated using TSA (Difco) with 0?1% sodium pyruvate (Fisher Scienti¢c, Fair Lawn, New Jersey, USA) (TSAP) (Leyer and Johnson 1992), and PALCAM (Difco) agar with PALCAM selective supplement (Dalynn Biological, Calgary, Canada). All plates were incubated at 301C for 48 h. The enumeration detection limit was 0?4 log cfu cm 2 . When numbers of bacteria dropped below the detection limit by direct plating, enrichment of samples was done. Brie£y, 1 ml of
Jerky processing to inactivate Listeria 549
the blended sample was transferred to 10 ml of UVM broth (Difco) and incubated at 301C for 24 h. Next, 0?1 ml of this culture was transferred to 10 ml of Fraser broth (Difco) and incubated at 351C for 48 h prior to streak plating onto PALCAM agar and incubation at 351C for 24 h for characteristic Listeria colonies (black). In addition to microbiological analyses, pH and water activity (Aw ) of beef jerky strips were determined at the each sampling interval. The pH was measured from samples used for microbiological analysis (25 ml BPW added and pummeled for 2 min) using a digital pH meter (Accumet 50; Fisher Scienti¢c, Houston, Texas, USA) with a glass pH electrode (Hanna Instruments, Ann Arbor, Michigan, USA). The Aw values of beef slices were determined using standardization methods as described in the 16th edn of Association of O⁄cial Analytical Chemists method no. 978.18 (Mulvaney 1998). One jerky slice was cut into small pieces to ¢t into the plastic container and measurement was done using a water-activity meter (Model D2101, Rotronic Instrument Corp., Huntington, New York, USA).
Statistical analysis Two independent replicates of the study were conducted. Microbiological data were converted to log cfu cm 2 based on the surface of fresh meat slices and evaluated using a 2 5 5 2 [(acid adaptation predrying treatments drying (sampling) times agar media, respectively)] factorial design. Data were analysed by analysis of variance for main (¢xed) e¡ects (acid adaptation, predrying treatment, drying time, and agar media) and four-way interactions between acid adaptation, predrying treatment, drying time, and agar media using the Statistical Analysis System (version 8?0, SAS Institute Inc., Cary, North Carolina, USA). Least-square means were separated using Fisher’s least signi¢cance di¡erence (LSD) test using the general linear models (GLMs) procedure of SAS. A signi¢cance level of 0?05 was used for all statistical analyses. Storage data were separately analysed. Data from each agar medium are presented separately (Figs 1^4) because the sizable volume would make combined presentation di⁄cult. Mean
counts of surviving bacterial populations between 0 and 7 h during drying were used to determine D-values by calculating the inverse of the slope of the linear regression line. Standard deviations of the pH and water-activity data were calculated.
Results Figs 1^4 show populations of L. monocytogenes during drying and storage of beef slices. Statistical analysis of the microbial data revealed that the main e¡ects of acid-adaptation, predrying treatment, drying time and agar media, and the interactions of acid adaptation predrying treatment drying time and predrying treatment drying time agar media were signi¢cant (Po0?05) for drying but not (PZ0?05) for storage data.
Temperature Changes in air temperature and meat surface temperatures during drying are shown in Fig. 5. During loading of the preheated dehydrator trays with the meat slices, the air temperature decreased from 601C to 401C at the beginning of drying, and then gradually increased back to 60711C within approximately 4 h. Surface temperature of meat slices from all trays reached 60721C within approximately 6 h. At the end of drying, surface temperatures of meat slices decreased to room temperature (c. 25^301C) within approximately 1 h.
E¡ect of agar media Signi¢cant di¡erences (Po0?05) in numbers of bacteria recovered on TSAP and PALCAM within each treatment were observed after 4 and 7 h of drying in MM, AATM and TWTM when the product was inoculated with non-adapted cultures. In products inoculated with acid-adapted cultures, signi¢cant di¡erences were observed in MM only after 4 h of drying. Bacterial counts recovered on two agar media from C and TM products were not significantly (PZ0?05) di¡erent during drying, but on day 15 during storage.These results indicate that MM, AATM and TWTM treatments
550 M. Calicioglu et al.
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+ Traditonal marinade (TWTM)
7 6
r2 0.99 0.96 0.93 0.95 0.99
D-value (h) 2.17 2.22 1.80 1.76 1.54
Log10 CFU/cm
2
5 4 3 2 1 0 -1 AI
0
2
4
6
8
10
Time (h)
(A)
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7
D-value (h) 2.11 2.15 1.66 1.52 1.51
2
r 0.99 0.96 0.90 0.91 0.88
6
Log10 CFU/cm 2
5 4 3 2 1 0 -1 AI
(B)
0
2
4
6
8
10
Time (h)
Figure 1. Survival of non-acid adapted L. monocytogenes as determined on TSAP (A) and PALCAM (B) during preparation (marination at 41C, 24 h) and drying (601C, 10 h) of beef jerky treated with various marinades (n = 4). AI: after inoculation; 0: after marination.Vertical bars are the standard deviation of the mean.
caused signi¢cant cellular injury to L. monocytogenes during drying while C and TM treatments resulted in only limited injury to cells.
E¡ect of predrying treatments Initial numbers of bacteria were signi¢cantly (Po0?05) reduced (1?0^1?7 log) after applica-
Jerky processing to inactivate Listeria 551
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7
D-value (h) 1.84 2.14 1.24 1.27 1.28
r2 0.90 0.94 0.91 0.84 0.87
6
Log10 CF U/cm
2
5 4 3 2 1 0 -1 AI
0
2
4
6
8
10
Time (h)
(A)
D- value (h) 1.90 2.10 1.20 1.20 1.27
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7 6
r2 0.92 0.98 0.80 0.81 0.81
Log10 CF U/ cm
2
5 4 3 2 1 0 -1 AI
(B)
0
2
4
6
8
10
Time (h)
Figure 2. Survival of acid-adapted L. monocytogenes as determined onTSAP (A) and PALCAM (B) during preparation (marination at 41C, 24 h) and drying (601C, 10 h) of beef jerky treated with various marinades (n = 4). AI: after inoculation; 0: after marination.Vertical bars are the standard deviation of the mean.
tion of AATM and TWTM and holding meat slices at 41C for 24 h, irrespective of acid-adaptation or agar media. Application of MM significantly reduced the populations of bacteria in products inoculated with non-adapted culture
only (Figs 1 and 2). Reductions ( 0?2 to 0?7 logs) in C and TM were not signi¢cant, irrespective of acid-adaptation. These results may indicate that immersing inoculated meat slices with L. monocytogenes in 5% acetic acid solution
552 M. Calicioglu et al.
3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditonal marinade (TWTM)
Log10 CFU/cm2
2
1
0
-1 0
10
20
30
40
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60
Day
(A)
3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1 0
10
20
(B)
30
40
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60
Day
Figure 3. Fate of surviving non-acid-adapted L. monocytogenes during aerobic storage at 251C for 60 days on beef jerky after treating with various marinades and drying at 601C for 10 h, as determined on TSAP (A) and PALCAM (B) (n = 4). 0 d: Bacterial counts at 10 h are included as day 0 counts for statistical analysis of the storage data.Vertical bars are the standard deviation of the mean.
followed by storage at 41C for 24 h are detrimental to L. monocytogenes, and potential cell detachment caused by Tween 20 was minimal or did not assist in increasing the subsequent killing e¡ect of acetic acid.
E¡ect of drying Irrespective of acid-adaptation or the recovery media used, major reductions (Po0?05) in bacterial counts occurred in all treatments
Jerky processing to inactivate Listeria 553
3 Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1
0
10
20
30
40
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60
Day
(A) 3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1 0
(B)
10
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60
Day
Figure 4. Fate of surviving acid-adapted L. monocytogenes during aerobic storage at 251C for 60 days on beef jerky after treating with various marinades and drying at 601C for 10 h, as determined on TSAP (A) and PALCAM (B) (n = 4). 0 d: Bacterial counts at 10 h are included as day 0 counts for statistical analysis of the storage data.Vertical bars are the standard deviation of the mean.
during the ¢rst 4 h of drying, while declines between 4 and 7 h of drying were smaller (Figs 1 and 2). No signi¢cant reduction in bacterial populations was found in any treatment inoculated with either acid- or non-
adapted cultures between 7 and 10 h of drying. In general, drying of MM, AATM, and TWTM products inoculated with both culture types resulted in signi¢cantly lower bacterial populations, as determined with both TSAP and
554 M. Calicioglu et al.
70
Temperature (°C)
60
50
40
30
Dehydrator air temperature Beef slice surface (bottom tray) Beef slice surface (middle tray) Beef slice surface (top tray)
20
10 0
2
4
6
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10
12
Drying time (h)
Figure 5. Mean (n = 4) temperatures of dehydrator air (middle hole air temperature) and the surface of beef slices during drying at 601C for 10 h using a home-type food dehydrator.
PALCAM, than did drying of C and TM treatments. There was no signi¢cant (PZ0?05) di¡erence in inactivation of acid-vs non-adapted inocula within individual treatments. However, numbers from AATM and TWTM treatments dropped below the detection limit (o 0?4 log cfu cm 2 ) of direct plating in products inoculated with acid-adapted cultures by 4 h of drying, whereas bacteria were still countable in products inoculated with non-adapted culture at 10 -h drying, indicating that acid-adapted cells may have been slightly more sensitive to these treatments. Bacterial counts in the C and TM treatments were not signi¢cantly different during drying, irrespective of agar media or acid-adaptation. Total log reductions in the numbers of bacteria in the products inoculated with nonadapted L. monocytogenes at the end of 10 h of drying as determined on non-selective (TSAP) and selective (PALCAM) media, were 3?8^4?6 for C, 4?0^4?3 for TM, 5?6^5?9 for MM, 5?9^6?2 for AATM and 5?9^6?2 for TWTM. Correspond-
ing reductions in log counts of bacteria on products inoculated with acid-adapted cells were 4?0^4?2 for C, 4?0^4?3 for TM, 5?7^6?6 for MM, 5?7^6?7 for AATM, and 6?3^6?7 for TWTM. D-values calculated based on bacterial counts determined on each of the two agar media between 0 and 7 h during drying are shown in Figs 1 and 2. Irrespective of inoculum acid-adaptation, MM, AATM and TWTM treatments resulted in smaller D-values than the control and TM indicating faster declines in the numbers of bacteria.
E¡ect of storage Populations of surviving bacteria in all treatments continued to decrease especially during the ¢rst 10^30 days of the 60 -day storage at ambient temperature (251C) (Figs 3 and 4).The signi¢cant declines observed only in treatments C and TM between 0 (after drying) and 15 days, resulted probably from the fact that high numbers of bacteria survived drying in these two treatments. In general, bacterial counts on
Jerky processing to inactivate Listeria 555
MM, AATM and TWTM products dropped below the detection limit earlier than those on C and TM products probably because of the lower bacterial counts on day 0 in these treatments in combination with lower pH and Aw . No signi¢cant di¡erence in counts between agar media was observed in any of the treatments during the entire storage period; this indicates that death of bacteria was occurring in all treatments during the entire storage period. The earliest complete elimination of the L. monocytogenes (enrichment negative) during product storage occurred on day 15 in AATMand TWTM-treated jerky, regardless of acidadaptation and in MM when the product was inoculated with non-adapted culture. By day 60, bacterial counts in all treatments were below the detection limit by direct plating on PALCAM and also on TSAP, with the exception of C when inoculated with non-adapted culture. At the end of storage, viable cells were recovered from C only, regardless of acidadaptation.
pH and Aw Overall, application of MM, AATM and TWTM reduced the pH of meat compared to that of the C and TM product (Table 1). No appreciable change in pH of the products was observed during drying or storage. Water activity of products decreased remarkably, as expected, during drying (Table 2). However, no notable di¡erence was found among treatments during storage. The ultimate Aw of the ¢nished products varied between 0?600 and 0?700. Slight £uctuations between sampling points were probably because of variation among slices.
Discussion The present study revealed that inactivation of L. monocytogenes during drying of beef jerky was a¡ected greatly by the type of predrying treatment. Although drying of jerky in all treatments reduced bacterial populations, the
Table 1. Mean (n = 4; standard deviation) pH values of beef jerky slices inoculated with acid- or nonadapted Listeria monocytogenes and subjected to various predrying marination treatments before drying at 601C for 10 h and storage at 251C for 60 days Inoculated with acid-adapted L. monocytogenes Steps Processing
Time
Ca
TMb
MMc
0h
5?43 (0?13) 5?62 (0?05) 5?56 (0?02) 5?63 (0?05)
5?25 (0?11) 5?41 (0?14) 5?58 (0?03) 5?58 (0?05)
4?44 (0?20) 4?73 (0?03) 4?70 (0?04) 4?84 (0?06)
4?34 (0?12) 4?63 (0?08) 4?59 (0?08) 4?66 (0?03)
5?65 5?61 4?82 (0?02) (0?03) (0?09) Day 30 5?63 5?46 4?71 (0?05) (0?09) (0?24) Day 60 5?66 5?54 4?75 (0?11) (0?16) (0?15)
4?69 (0?04) 4?52 (0?13) 4?73 (0?13)
4h 7h 10 h Storage
a
Day 15
AATMd TWTMe
Inoculated with non-acid adapted L. monocytogenes C
TM
MM
AATM
TWTM
4?29 (0?12) 4?58 (0?11) 4?58 (0?04) 4?65 (0?01)
5?69 (0?03) 5?78 (0?04) 5?74 (0?12) 5?70 (0?06)
5?57 (0?02) 5?75 (0?08) 5?68 (0?12) 5?62 (0?09)
4?61 (0?07) 4?77 (0?09) 4?77 (0?15) 4?83 (0?06)
4?62 (0?08) 4?84 (0?07) 4?91 (0?03) 4?88 (0?12)
4?61 (0?11) 4?85 (0?03) 4?80 (0?04) 4?72 (0?11)
4?72 (0?04) 4?63 (0?11) 4?75 (0?11)
5?48 (0?14) 5?41 (0?05) 5?55 (0?06)
5?39 (0?16) 5?35 (0?11) 5?53 (0?09)
4?54 (0?32) 4?55 (0?04) 4?76 (0?07)
4?51 (0?18) 4?61 (0?18) 4?72 (0?18)
4?40 (0?24) 4?61 (0?14) 4?69 (0?16)
No predrying treatment or marinade prior to refrigeration of inoculated slices at 41C for 24 h and drying. Marinated with TM (pH 4?3), held at 41C for 24 h, and then dried. c Marinated with modi¢ed marinade (1?2% lactate, 9% acetic acid, and soy sauce w/ 5% ethanol) ( pH. 3?0), held at 41C for 24 h, and then dried. d Dipped into 5% acetic acid solution (pH 2?5) for 10 min at ambient temperature, drained for 2 min, and then marinated with TM. e Dipped into 1% Tween 20 (pH 6?6) for 15 min, and then 5% acetic acid solution for 10 min at ambient temperature, followed by marination with TM. b
556 M. Calicioglu et al.
Table 2. Mean (n = 4; standard deviation) Aw values of beef jerky slices inoculated with acid- or nonadapted L. monocytogenes and subjected to various predrying marination treatments before drying at 601C for 10 h and storage at 251C for 60 days Inoculated with acid-adapted L. monocytogenes Steps Processing
Time
Ca
TMb
MMc
0h
0?965 (0?005) 0?911 (0?018) 0?828 (0?010) 0?659 (0?016)
0?956 (0?003) 0?793 (0?085) 0?697 (0?107) 0?642 (0?023)
0?945 (0?001) 0?798 (0?023) 0?721 (0?006) 0?481 (0?044)
0?949 (0?006) 0?851 (0?074) 0?638 (0?033) 0?684 (0?034)
0?645 0?610 0?603 (0?009) (0?050) (0?011) Day 30 0?650 0?657 0?657 (0?007) (0?061) (0?002) Day 60 0?664 0?646 0?627 (0?014) (0?032) (0?021)
0?621 (0?049) 0?625 (0?003) 0?615 (0?021)
4h 7h 10 h Storage
Day 15
AATMd TWTMe
Inoculated with non-acid adapted L. monocytogenes C
TM
MM
AATM TWTM
0?950 (0?010) 0?878 (0?031) 0?786 (0?096) 0?579 (0?097)
0?961 (0?002) 0?900 (0?008) 0?747 (0?023) 0?697 (0?081)
0?948 (0?004) 0?904 (0?018) 0?804 (0?095) 0?642 (0?064)
0?955 (0?002) 0?830 (0?013) 0?603 (0?087) 0?567 (0?027)
0?623 (0?018) 0?684 (0?002) 0?643 (0?004)
0?667 0?633 0?597 0?613 0?668 (0?016) (0?027) (0?083) (0?021) (0?024) 0?652 0?624 0?606 0?643 0?662 (0?003) (0?001) (0?008) (0?061) (0?031) 0?666 0?602 0?575 0?636 0?658 (0?020) (0?066) (0?010) (0?065) (0?011)
0?950 (0?004) 0?793 (0?124) 0?690 (0?011) 0?658 (0?099)
0?952 (0?005) 0?889 (0?068) 0?774 (0?154) 0?618 (0?037)
a
No pre-drying treatment or marinade prior to refrigeration of inoculated slices at 41C for 24 h and drying. Marinated with TM (pH 4?3), held at 41C for 24 h, and then dried. c Marinated with modi¢ed marinade (1?2% lactate, 9% acetic acid, and soy sauce w/ 5% ethanol) (pH. 3?0), held at 41C for 24 h, and then dried. d Dipped into 5% acetic acid solution (pH 2?5) for 10 min at ambient temperature, drained for 2 min, and then marinated with TM. e Dipped into 1% Tween 20 (pH 6?6) for 15 min, and then 5% acetic acid solution for 10 min at ambient temperature, followed by marination with TM. b
modi¢ed marinades MM, AATM and TWTM applied as predrying treatments led to greater levels of reduction during drying. Survival of the pathogen during drying was not signi¢cantly di¡erent between control and traditionally marinated (TM) jerky, in the present study, indicating no additional antimicrobial e¡ect from TM. Although TM contains Worcestershire sauce, it was not acidic enough (pH 4?3) to cause an appreciable reduction in the pH of the TM product compared to control products, given the limited volume used per beef slice. The e¡ectiveness of TM in facilitating reduction in numbers of L. monocytogenes and other pathogens during drying was studied by other researchers. Harrison and Harrison (1996) reported that drying traditionally marinated whole muscle jerky at 601C for 10 h was su⁄cient to deliver 45?0 -log units reduction of E. coli O157:H7, L. monocytogenes, and S. typhimurium, as determined with selective agar media. In a more comprehensive study, Harrison et al. (2001) compared the e¡ectiveness of TM, oven-
heating at 711C before drying, boiling in marinade prior to drying, and post-drying heating of traditionally marinated jerky at 57?31C for 10 min in reducing numbers of E. coli O157:H7, L. monocytogenes, and Salmonella on whole muscle beef jerky during drying at 601C for 10 h. Their results showed that heating and boiling before drying reduced the numbers of all three pathogens, prior to drying, below the detection limit on selective agar media (MOX for Listeria), indicating an immediate action. At the end of drying, total reductions from all treatments were equal to or greater than 5?8, 3?9, and 4?6 -log units for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively, even with TM. Consumer acceptability of product heated after drying or boiled in marinade before drying was signi¢cantly higher than that for product heated before drying. The authors concluded that oven-heating jerky after drying resulted in safer and acceptable jerky product. In the present study, C and TM resulted in lower bacterial reductions
Jerky processing to inactivate Listeria 557
compared to use of MM, AATM or TWTM during drying, regardless of acid adaptation. In other words, at the end of drying, signi¢cantly higher numbers of bacteria were recovered from C and TM jerky on both TSAP and PALCAM than from jerky of other treatments. These results might indicate that predrying treatments MM, AATM and TWTM can improve the e¡ectiveness of drying on survival of L. monocytogenes further than TM alone. It has been reported that simultaneous exposure of bacteria to di¡erent stress factors requires increased energy consumption and leads bacteria to cellular death through metabolic exhaustion and disturbed homeostasis (Leistner 2000). Barker and Park (2001) reported that combinations of osmotic stress (NaCl, sucrose or glycerol), ethanol (5%), and high acid (pH 3?0, HCl) or 50 mmol concentrations of food-grade acid salts (including DL -lactate) provided reductions of stationary phase L. monocytogenes up to 5 logs in 4 min. Their results further indicated that such combination of organic acids, ethanol, and low pH killed the cells by damaging the bacterial cell membrane. This phenomenon was modi¢ed and adopted as a predrying treatment (modi¢ed marinade or MM) in the present study, by using commercially available soy sauce containing approximately 5% ethanol, and adding 2% of a 60% sodium lactate preparation, and 9% acetic acid to TM.The volume of marinade solution was also increased by using double the volume of soy sauce and Worcestershire sauce to deliver a higher marinade solution per slice than TM. The MM used in the present study was shown to be signi¢cantly more e¡ective in reducing the populations of L. monocytogenes cells on beef slices during drying compared to the use of TM and C, regardless of acid-adaptation. These ¢ndings indicated that combinations of lactate, ethanol and acetic acid might have a potential to be used in destruction of pathogenic bacteria in processed foods. It is known that bacterial cells attached to the surface of a product (such as meat) become more resistant to stress factors (such as heat) than non-attached cells (Humphrey et al. 1997). Another study showed that prespraying beef carcasses inoculated with high levels of E. coli 0157:H7 with 5% Tween 20 followed by
spraying with 2% lactic acid resulted in significantly higher reduction of the pathogen compared to spraying with lactic acid alone or water (Calicioglu et al. 2002). It has been speculated that Tween 20 may loosen cells or prevent cellular attachment on the meat surface via its surfactant and hydrophobic e¡ects, thus making the cells more vulnerable to the e¡ect of subsequent acid exposure. This proposed carcass decontamination method was applied as a predrying treatment in the present study (TWTM) prior to acetic acid dip and marinating with a TM recipe. Tween 20 (polysorbate 20, or polyoxyethylene-20 -sorbitan monolaurate) is permitted to be used in food as adjuvant (Code of Federal Regulations 2001). In the present study, there was no signi¢cant di¡erence in destruction of bacteria during drying between AATM and TWTM, regardless of acid adaptation, sampling time or recovery media. Both treatments were signi¢cantly more e¡ective than C and TM, even before drying. Lesser resistance of L. monocytogenes to acid conditions compared to E. coli O157:H7 (Roering et al. 1999, Samelis et al. 2001b) might explain this observation. In general, numbers of bacteria continued to decline in all treatments until 7 h of drying and then followed a much slower reduction rate, or did not change. This decelerated reduction in counts has been described as a ‘‘tailing e¡ect’’. In the present study, the tailing e¡ect was seen in treatments involving MM, AATM and TWTM, particularly, as determined by selective agar media, between 4 or 7 and 10 h of drying. Buchanan et al. (1994) investigated nonthermal inactivation of L. monocytogenes and reported that the tailing e¡ect was not because of the presence of a more resistant subpopulation. Their results revealed that inactivation rates of the pathogen in liquid media, as affected by pH and sublethal temperatures (up to 401C), were independent of initial population density. In contrast, as mentioned, preculturing of L. monocytogenes in acidic environment or in BHI broth with 0?2^0?5% glucose (acid-adaptation) increased the general acid resistance of the pathogen (Buchanan et al. 1994). There has been additional evidence that acid-adaptation of L. monocytogenes enhances its survival in acidic foods and that it may
558 M. Calicioglu et al.
increase cross-protection to other type of stresses (Gahan et al. 1996, Lou and Yousef 1997, Saklani-Jusforgues et al. 2000, Francis and O’Beirne 2001). In the present study, however, no enhanced survival of acid-adapted L. monocytogenes was observed during drying in any treatment. Similar results have been reported for other acid-adapted pathogens, such as Salmonella and Escherichia coli O157:H7, exposed subsequently to acid or additional stresses (e.g. heat, osmotic stress) during a food process (Dickson and Kunduru 1995, Ryu et al. 1999, Riordan et al. 2000). For example, Ryu et al. (1999) compared the survival of acidadapted, non-adapted and acid-shocked cells of E. coli O157:H7 in dried beef powder during storage.They reported that there was no significant di¡erence in survival of these cell types of the pathogen. The authors then concluded that acid-adaptation did not result in crossprotection against dehydration or osmotic stress. This di¡erence might be the result of use of multiple hurdles involved in the meatdrying process such as high acid, temperature, and low Aw as well as the in£uence of acidadaptation. It might be speculated that level of cross-protection from acid-adaptation is dependent on the presence and severity of other stress factors as a function of time. It seems, in the present study, that acid (e.g. rpH 4?8), heat (i.e.Z601C) and low water activity (o0?80) may provide a combination of hurdles able to overcome any resistance provided by acid-adaptation. In addition, the physiological state of the cells during treatment may in£uence their resistance or cross-protection behavior (McClements et al. 2001). In conclusion, results of the present study indicated that acid-adapted cells of L. monocytogenes did not exhibit any increased resistance to the drying process compared to that of nonadapted cells. Our results also revealed that using food-grade chemicals as predrying treatments (MM, AATM, and TWTM) improved the e¡ectiveness of the meat-drying process for inactivating L. monocytogenes, compared to the traditional jerky-making process. Studies of the e¡ects of such treatments on the sensory attributes of the ¢nal product are in progress. Under the conditions described in the present study, use of MM, AATM or TWTM in the
jerky-making process and storing the dried products for 15 days at ambient temperature, or use of TM and then storing for 60 days prior to consumption, should inactivate 6^7 logs of the pathogen.
Acknowledgements This project was funded by USDA-CSREES and by the Colorado Agricultural Experiment Station. We thank Excalibur Seasoning Company, and Heller Seasoning and Ingredients Inc., for providing irradiated spices.
References Albright, S. N. D. (2000) Evaluation of processes to destroy Escherichia coli O157:H7 in whole muscle home dried beef jerky. Ph.D. Dissertation. Colorado State University. Andress, E. L. and Harrison, J. A. (1999) So easy to preserve. 4th Edn. Athens, GA, USA, Cooperative Extension Service, University of Georgia. Barker, C. and Park, S. F. (2001) Sensitization of Listeria monocytogenes to low pH, organic acids and osmotic stress by ethanol. Appl. Environ. Microbiol. 67, 1594^1600. Buchanan, R. L., Golden, M. H., Whiting, R. C., Philips, J. G. and Smith, J. L. (1994) Non-thermal inactivation models for Listeria monocytogenes. J. Food Sci. 59, 179^188. Calicioglu, M., Kaspar, C. W., Buege, D. R. and Luchansky, J. B. (2002) E¡ectiveness of spraying with Tween 20 and lactic acid to decontaminate inoculated Escherichia coli O157:H7 and indigenous E. coli biotype I on beef. J. Food Prot. 65, 26^32. Centers for Disease Control and Prevention. (1995) Outbreak of Salmonellosis associated with beef jerky F New Mexico. Morb. Mortal.Wkly Rep. 44, 785^788. Code of Federal Regulations, Title 21 (Food and Drugs) (2001) Food Additives Permitted for Direct Addition to Food for Human Consumption, chapter I, part 172; Synthetic Flavoring Substances and Adjuvants (172.515). Washington, DC., The Government Printing O⁄ce (GPO). Dickson, J.S. and Kunduru, M.R. (1995) Resistance of acid-adapted Salmonellae to organic acid rinses on beef. J. Food Prot. 58, 973^976. Francis, G. A. and O’Beirne, D. (2001) E¡ects of acid adaptation on the survival of Listeria monocytogenes on modi¢ed atmosphere packaged vegetables. Int. J. Food Sci. Technol. 36, 477^487.
Jerky processing to inactivate Listeria 559
Gahan, G. M. C., O’Driscoll, B. and Hill, C. (1996) Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Appl. Environ. Microbiol. 62, 3128^3132. Gailani, M. B. and Fung, D. Y. C. (1986) Critical review of water activities and microbiology of drying of meats. Crit. Rev. Food Sci. Nutr. 25, 159^183. Gould, G. W. (2001) New processing technologies: an overview. Proc. Nutr. Soc. 60, 463^474. Harrison, J. A. and Harrison, M.A. (1996) Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium during preparation and storage of beef jerky. J. Food Prot. 59, 1336^1338. Harrison, J. A., Harrison, M. A., Rose, R. A. and Shewfelt, R. A. (2001) Home-style beef jerky: e¡ect of four preparation methods on consumer acceptability and pathogen inactivation. J. Food Prot. 64, 1194^1198. Humphrey,T. J.,Wilde, S. J. and Rowbury, R. J. (1997) Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue. Lett. Appl. Microbiol. 25, 265^268. Keene,W. E., Sazie, E., Kok, J., Rice, D. H., Hancock, D. D., Balan,V. K., Zhao, T. and Doyle, M. P. (1997) An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat. JAMA. 227, 1229^1231. Leistner, L. (2000) Basic aspects of food preservation by hurdle technology. Int. J. Food Microbiol. 55, 181^186. Levine, P., Rose, B., Green, S., Ransom, G. and Hill, W. (2001) Pathogen testing of ready-to-eat meat and poultry products collected at federally inspected establishments in the United States, 1990 to 1999. J. Food Prot. 64, 1188^1193. Leyer, G. J. and Johnson, E. A. (1992) Acid adaptation promotes survival of Salmonella spp. in cheese. Appl. Environ. Microbiol. 58, 2075^2080. Lou, Y. and Yousef, A. E. (1997) Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors. Appl. Environ. Microbiol. 63, 1252^1255. McClements, J. M., Patterson, M. F. and Linton, M. (2001) The e¡ect of growth stage and growth temperature on high hydrostatic pressure inactivation of some psychrotrophic bacteria in milk. J. Food Prot. 64, 514^522. Mulvaney,T. R. (1998) Vegetable products, processed. In O⁄cial Methods of Analysis of AOAC Interna-
tional, 16th edn (Ed P. Cunnif) Chap. 42, pp. 1^2. Arlington, VA, USA, Association of O⁄cial Analytical Chemists. Riordan, D. C. R., Du¡y, G., Sheridan, J. J., Whiting, R. C., Blair, I. S. and McDowell, D. A. (2000) E¡ect of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni. Appl. Environ. Microbiol. 66, 1726^1729. Roering, A. M., Luchansky, J. B., Ihnot, A. M., Ansay, S. E., Kaspar, C. W. and Ingham, S. C. (1999) Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric £uid. Int. J. Food Microbiol. 46, 263^269. Ryu, J.-H., Deng,Y. and Beuchat, L. R. (1999) Survival of Escherichia coli O157:H7 in dried beef powder as a¡ected by water activity, sodium chloride content and temperature. Food Microbiol. 16, 309^316. Saklani-Jusforgues, H., Fontan, E. and Goossens, P. L. (2000) E¡ect of acid adaptation on Listeria monocytogenes survival and translocation in a murine intragastric infection model. FEMS Microbiol. Lett. 193, 155^159. Samelis, J., Sofos, J. N., Kendall, P. A. and Smith, G. C. (2001a) In£uence of natural microbial £ora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination £uids. Appl. Environ. Microbiol. 67, 2410^ 2420. Samelis, J., Sofos, J. N., Kendall, P. A. and Smith, G. C. (2001b) Fate of Escherichia coli O157:H7, Salmonella typhimurium DT 104, and Listeria monocytogenes in fresh meat decontamination £uids at 4 and 10o C. J. Food Prot. 64, 950^957. Samelis, J., Sofos, J. N., Kendall, P. A. and Smith, G. C. (2001c) Behavior of acid-adapted L. monocytogenes in meat decontamination washings. In Proceedings of the 47th International Congress of Meat Science and Technology Vol. II. pp. 54^55. Krakow, Poland. United States Code. (2002) Inspection requirements; adulteration and misbranding. Title 21, Meat Inspection 601(m). United States Department of Agriculture-Food Safety and Inspection Service. (1998) Consumer education and information: http://www.fsis.usda.gov/OA/pubs/jerky/html
doi:10.1006/yfmic.510
ORIGINAL ARTICLE
Destruction of acid- and non-adapted Listeria monocytogenes during drying and storage of beef jerky Mehmet Calicioglu1, John N. Sofos 1; *, John Samelis1, Patricia A. Kendall2 and Gary C. Smith1
Presence of Listeria monocytogenes in ready-to-eat meat products is not desired and strictly regulated in the US. Inactivation of acid- and non-adapted L. monocytogenes inoculated on beef slices was studied during drying and storage of jerky formulated with modi¢ed marinades. The inoculated (¢ve-strain composite, c. 6?2 log cfu cm 2) slices were subjected to marinades (41C, 24 h) prior to drying (601C for 10 h) and aerobic storage (251C for 60 days). The predrying marinade treatments tested were, ¢rst, no treatment, control (C); second, traditional marinade (TM); third, double amount of TM modi¢ed with 1?2% sodium lactate, 9% acetic acid, and 68% soy sauce containing 5% ethanol (MM); fourth, dipping into 5% acetic acid for 10 min and then applying theTM (AATM); and ¢fth dipping into 1% Tween 20 for 15 min and then into 5% acetic acid for 10 min followed by theTM (TWTM). Bacterial survivors on beef slices were determined during drying and storage using tryptic soy agar with 0?1% pyruvate (TSAP), and PALCAM agar. Results indicated that drying reduced bacterial populations in the order of pre-drying treatments TWTM (5?9^6?3 log cfu cm 2 in 10 h)ZAATMZMM4TMZC (3?8 4?6 log cfu cm 2 in 10 h). No signi¢cant (PZ0?05) di¡erence was found in inactivation of acidadapted and non-adapted inocula within individual treatments. Bacterial populations dropped below the detection limit ( 0?4 log cfu cm 2) as early as 4 h during drying or remained detectable even after 60 days of storage depending on acid-adaptation, predrying treatment, and agar media. These results indicated that acid-adaptation may not increase resistance to microbial hurdles involved in jerky processing and that use of modi¢ed marinades may improve the e¡ectiveness of drying in inactivating L. monocytogenes. # 2002 Elsevier Science Ltd. All rights reserved. Received: 28 February 2002
Introduction Dried products, such as jerky, have been considered as one of the safest food groups for humans because their manufacture involves hurdles to microbial survival or growth. Such
*Corresponding author. Fax: +1 970 491 0278. E-mail: [email protected] 0740 -0020/02/060545+15 $35.00/0
hurdles include low water activity (o0?85), drying temperature, and use of preservatives such as salt, organic acid and sodium nitrite, depending on the composition of the marination mixture (Gailani and Fung 1986). However, a recent report (Levine et al. 2001) by the Food Safety and Inspection Service of the United States Department of Agriculture (FSIS/ USDA) indicated that for the period 1990^ 1999, cumulative prevalence of Salmonella and r 2002 Elsevier Science Ltd. All rights reserved.
1 Department of Animal Sciences, Center for Red Meat Safety, Colorado State University, Fort Collins, Colorado 80523, USA 2 Department of Food Science and Nutrition, Colorado State University, Fort Collins, Colorado 80523, USA
546 M. Calicioglu et al.
Listeria monocytogenes in jerky produced in federally inspected plants was 0?31% and 0?52%, respectively. Currently, FSIS applies a‘zero-tolerance’ policy for L. monocytogenes in ready-toeat meat products including jerky. Products that are not in compliance with this policy are considered to being adulterated under the provisions of United States Federal Meat Inspection Act (21 US Code, 601 (m)). The recent Salmonella and Escherichia coli O157:H7 outbreaks linked to jerky (CDC 1995, Keene et al. 1997) have renewed interest in evaluating the e⁄cacy of jerky processing, especially when prepared in home-type dehydrators, for inactivating pathogens (Albright 2000, Harrison and Harrison 1996, Harrison et al. 2001, Keene et al. 1997). As a response to the problem, the USDA/FSIS (1998) has recommended cooking meat to 71?11C before drying to eliminate the risk of pathogen survival. However, traditional jerky drying (601C, 10 h) after marination, irrespective of predrying heating, has been shown to be e¡ective in reducing (e.g., 5 -log unit reduction) E. coli O157:H7, Salmonella typhimurium and L. monocytogenes (Harrison and Harrison 1996; Harrison et al. 2001). In contrast, Keene et al. (1997) reported that drying at r63o C was not a reliable method for eliminating E. coli O157:H7 in marinated (pH 4?2) whole muscle venison jerky. Preheating of meat and/or drying of jerky at high temperatures for extended periods of time may result in a product that di¡ers texturally from traditional jerky and, thus, it may have reduced consumer acceptability. There is a need for development of new food preservation strategies with the objective of inactivating bacteria while avoiding severe treatments that change desired characteristics of the food, because most traditional food preservation methods (e.g. chilling, freezing, drying) slow down or inhibit growth of bacteria rather than inactivating them (Gould 2001). Leistner (2000) indicated that preservation methods/ technologies that simultaneously or sequentially expose bacteria to multiple stress factors (hurdles) lead bacteria to metabolic exhaustion or homeostatic disturbance followed by cellular death. Thus, employing intervention strategies that sensitize bacteria attached to meat slices via predrying treatments to drying at
mild temperatures (e.g. 60o C) can be a viable option to avoid severe heat treatments. Examples of such interventions might be weakening bacterial attachment by use of surfactants followed by dipping in organic acid solution prior to marination and drying or designing new marinade solutions that can disturb or damage bacterial homeostasis. These marinades may provide residual antimicrobial e¡ects during storage against possible post-processing contamination. There is some evidence that acid-adaptation of L. monocytogenes may enhance its survival in acidic foods as well as increase cross-protection to other type of stresses (Buchanan et al. 1994, Gahan et al. 1996, Lou and Yousef 1997, Saklani-Jusforgues et al. 2000, Francis and O’Beirne 2001).To date, it is not known whether acid-adapted pathogenic bacterial cells survive better than non-acid-adapted cells during the jerky-making process. Therefore, the objective of the present study was to evaluate the e¡ectiveness of various chemical-based predrying treatments (modi¢ed marinades) in enhancing destruction of acid- or non-adapted L. monocytogenes cells during drying and storage of whole muscle beef jerky.
Materials and Methods Bacterial strains used and preparation of inoculum A ¢ve-strain composite of L. monocytogenes was used for inoculating beef strips. These strains were LM101 (sausage isolate, serotype 4b), LM103 (sausage isolate, serotype 1a), N7143 (meat isolate, serotype 3a), N-7144 (meat isolate, serotype 1/2a), and TB2000 (turkey breast isolate, unknown serotype). Each strain was propagated (301C, 24 h) and maintained on tryptic soy agar (TSA, Difco Laboratories, Detroit, Michigan, USA) slants at 41C. Strains were subcultured monthly. The cultures were activated by transferring a loopful of each strain into 9 ml of tryptic soy broth (TSB, Difco) and incubating at 301C for 24 h. Acid-adaptation was induced by preculturing strains in media with glucose, a condition previously reported to result in increased acid tolerance as
Jerky processing to inactivate Listeria 547
compared to L. monocytogenes cultures grown without glucose (Buchanan et al. 1994). More speci¢cally, a 0?1-ml portion of activated culture of each strain was transferred into 9 -ml tubes of glucose-free TSB (TSB-G) (BBL, Becton Dickinson Co., Cockeysville, Maryland, USA) for non-acid-adapted cells (Buchanan et al. 1994) and in glucose-free TSB with 1% glucose (Sigma, St. Louis, Missouri, USA) added (TSB+G) for acid-adapted cells (Samelis et al. 2001c). Addition of 1% glucose was aimed at enhancing acid-adaptation of TSB+G cultures, although 0?3% glucose added to brain^heart infusion (BHI) broth, or even 0?2% or 0?25% glucose as part of the normal formulation of BHI and TSB, respectively, have been found su⁄cient to deliver signi¢cant increases in acid tolerance compared to TSB-G cultures of L. monocytogenes (Buchanan et al. 1994, Samelis et al. 2001a). The challenge method used to assess acid tolerance was described previously (Samelis et al. 2001a). Con¢rmatory experiments during previous studies (Samelis et al. 2001c) have shown that with 1% glucose added to TSB-G, the pH values of the resulting acid-adapted cultures (301C, 24 h) of L. monocytogenes strains N-7144 and N7143 were 4?4^4?5 as compared with pH values of 6?6^6?7 of their non-adapted counterparts grown in TSB-G. This induced acid-adaptation via glucose fermentation by L. monocytogenes provided signi¢cant protection to the pathogen following a 2 -h exposure to acidi¢ed TSB with lactic acid, pH 3?5 (e.g. 1?0^1?3 log population reductions) as compared to the corresponding non-adapted populations (Z3?9 -log reductions) (unpublished data). Thus, the TSB+G and TSB-G cultures of L. monocytogenes strains used in this study were acidand non-adapted, respectively. After incubation at 301C for 22^24 h, individual cultures were combined in a sterile tube for centrifuging at 6000 rpm (2900 g) (Eppendorf, model 5402) for 30 min at 41C. The resulting pellet was washed once with 0?1% phosphatebu¡ered saline (PBS, Sigma) to remove residual organic material, recentrifuged, and then resuspended in PBS to a ¢nal volume of 100 ml. The average cell concentration in the resulting composite inoculum was 7?0 log cfu ml 1 .
Preparation of meat slices Vacuum-packaged and frozen (^181C) beef inside rounds (o3 months) were purchased from the Colorado State University Meat Science Laboratory (Fort Collins, Colorado, USA). Following thawing at 41C overnight, inside rounds were sliced at 0?6 -cm thickness using a food slicer (model 610, Hobart Corp., Troy, Ohio, USA) and cut into pieces of 8?7 4?0 cm2 using a plastic template and knife. Approximately, 100 slices (2?2 kg) of meat were vacuum-packaged and kept frozen at ^181C until use (1 ^ 3 weeks).
Inoculation procedure Frozen beef slices were thawed at 41C for 24 h, placed on plastic trays and inoculated under a laminar-£ow hood. Portions of 0?5 ml of the L. monocytogenes inoculum were placed on the upper surface of each slice and spread onto the entire surface area using a sterile bent glass rod. Bacteria were allowed to attach to the meat surface for 15 min at ambient temperature. The slices were then £ipped over and the other side was inoculated following the same procedure. The resulting level of inoculum was approximately 6?2 log cfu cm 2 .
Predrying treatments They included ¢rst, control, no treatment (C); second, marination with traditional marinade (pH 4?3) (TM); third, increased (double the amount) marination with modi¢ed marinade (pH 3?0) (MM), fourth, dipping into 5% acetic acid solution followed by traditional marination (AATM); and ¢fth, sequential 15 -min immersion in 1% Tween 20 solution (polyoxyethylene-20 -sorbitan monolaurate) (pH 6?6), then 10 -min immersion in 5% acetic acid solution (pH 2?5) followed by marination with TM (TWTM). The TM was prepared for 1?0 kg of meat (Andress and Harrison 1999) as follows: 60 ml soy sauce (Kikkoman Foods, Walworth, Wisconsin, USA), 15 ml Worcestershire sauce (Heinz, Pittsburgh, Pennsylvania,USA), 0?6 g black pepper (Heller Seasoning and Ingredients Inc., Chicago, Illinois, USA), 1?25 g garlic powder (Excalibur Seasoning Co. Ltd. Pekin, Illinois, USA), 1?5 g onion powder (Excalibur),
548 M. Calicioglu et al.
4?35 g old hickory smoked salt (Tone Brothers Inc., Ankeny, Iowa, USA). In the present study, 30 ml of this marinade was used for 450 g of inoculated meat. Marinade was spread manually on beef slices and then the slices were manually mixed to cover the entire surface area using £ame-sterilized forceps. The modi¢ed marinade was prepared for 1?0 kg of meat as follows: 120 ml of ‘‘milder soy sauce’’ (Kikkoman) containing approximately 4?7^5?0% ethanol as preservative, 30 ml of Worcestershire sauce, 0?6 g black pepper, 1?25 g garlic powder, 1?5 g onion powder, 4?35 g smoke-£avored salt, 3?6 ml of 60% food-grade sodium-L -lactate preparation (Purac Inc., Lincolnshire, Illinois, USA), and 16 ml of glacial acetic acid (Mallinckrodt Baker Inc., Paris, Kentucky) to adjust to pH 3?0. A 60 -ml portion of this marinade solution (double that of TM) was spread on 450 g of beef, and mixed to cover surfaces of meat slices. For the fourth treatment (AATM), meat slices were dipped into 5% (v/v) acetic acid solution prepared using glacial acetic acid at ambient temperature for 10 min (450 ml per 450 g of meat). These slices were drained for 2 min to remove excessive £uid using an empty dehydrator tray and placed on a tray covered with aluminum foil followed by marination with TM. For the ¢fth treatment (TWTM), meat slices were dipped into 1% (v/v) Tween 20 (Fisher Scienti¢c Inc., Fair Lawn, New Jersey, USA) solution (450 ml per 450 g of meat) for 15 min at ambient temperature in a 1000 ml glass container. These slices were drained for 2 min, and then the same steps as were used for treatment AATM were followed. Black pepper, garlic and onion powder were irradiated by their manufacturers. Following each treatment, trays containing the inoculated and marinated slices were covered with aluminum foil and held at 41C for 24 h prior to drying.
Drying After the 24 h of refrigeration, the inoculated and treated meat slices were dried at 601C for 10 h in a home-type dehydrator (American Harvest Gardenmaster, model FD-1000, Nesco, Chaska, Minnesota, USA; two appliances used). The dehydrators were cylindrical in shape and were composed of a base unit and
three drying trays. The dehydrator base unit generated hot air, which ventilated upward through the sides and a hole in the middle of the trays. The target temperature was based on the air temperature measurement taken from the middle hole of the dehydrator. The dehydrators with empty trays were preheated for approximately 20 min to 601C (1401F). The empty trays were then replaced with other trays preloaded with meat slices. During drying, the temperature of the dehydrator through the middle hole and the surface temperature of meat slices on each of the bottom, middle, and top trays were monitored using thermocouples (Type K beaded probes, Pico Technology Ltd. Cambridge, UK) and real-time data recording software (Pico Technology). After drying, the jerky strips were held in the dehydrators overnight as recommended by Andress and Harrison (1999), and then placed into 24 -oz WhirlPak sterile plastic bags (Nasco, Fort Atkinson, Wisconsin, USA) for storage at ambient temperature (25711C).
Analysis Two samples (1 slice sample 1 ) per treatment were aseptically transferred into sterile-plastic bags (Nasco, Modesto, California, USA) at each sampling interval. These intervals included after inoculation, and 0 (after marination and 24 h refrigeration), 4, 7 and 10 h during drying for each treatment, and days 15, 30 and 60 during storage. A 25 ml portion of 0?1% sterile bu¡ered peptone water (BPW) (Difco) was added to each sample bag prior to pummeling for 2 min at ambient temperature. Serial decimal dilutions were made using 9 -ml BPW tubes and 0?1-ml portions were surface plated onto each of duplicate plates of each agar medium. Bacteria were enumerated using TSA (Difco) with 0?1% sodium pyruvate (Fisher Scienti¢c, Fair Lawn, New Jersey, USA) (TSAP) (Leyer and Johnson 1992), and PALCAM (Difco) agar with PALCAM selective supplement (Dalynn Biological, Calgary, Canada). All plates were incubated at 301C for 48 h. The enumeration detection limit was 0?4 log cfu cm 2 . When numbers of bacteria dropped below the detection limit by direct plating, enrichment of samples was done. Brie£y, 1 ml of
Jerky processing to inactivate Listeria 549
the blended sample was transferred to 10 ml of UVM broth (Difco) and incubated at 301C for 24 h. Next, 0?1 ml of this culture was transferred to 10 ml of Fraser broth (Difco) and incubated at 351C for 48 h prior to streak plating onto PALCAM agar and incubation at 351C for 24 h for characteristic Listeria colonies (black). In addition to microbiological analyses, pH and water activity (Aw ) of beef jerky strips were determined at the each sampling interval. The pH was measured from samples used for microbiological analysis (25 ml BPW added and pummeled for 2 min) using a digital pH meter (Accumet 50; Fisher Scienti¢c, Houston, Texas, USA) with a glass pH electrode (Hanna Instruments, Ann Arbor, Michigan, USA). The Aw values of beef slices were determined using standardization methods as described in the 16th edn of Association of O⁄cial Analytical Chemists method no. 978.18 (Mulvaney 1998). One jerky slice was cut into small pieces to ¢t into the plastic container and measurement was done using a water-activity meter (Model D2101, Rotronic Instrument Corp., Huntington, New York, USA).
Statistical analysis Two independent replicates of the study were conducted. Microbiological data were converted to log cfu cm 2 based on the surface of fresh meat slices and evaluated using a 2 5 5 2 [(acid adaptation predrying treatments drying (sampling) times agar media, respectively)] factorial design. Data were analysed by analysis of variance for main (¢xed) e¡ects (acid adaptation, predrying treatment, drying time, and agar media) and four-way interactions between acid adaptation, predrying treatment, drying time, and agar media using the Statistical Analysis System (version 8?0, SAS Institute Inc., Cary, North Carolina, USA). Least-square means were separated using Fisher’s least signi¢cance di¡erence (LSD) test using the general linear models (GLMs) procedure of SAS. A signi¢cance level of 0?05 was used for all statistical analyses. Storage data were separately analysed. Data from each agar medium are presented separately (Figs 1^4) because the sizable volume would make combined presentation di⁄cult. Mean
counts of surviving bacterial populations between 0 and 7 h during drying were used to determine D-values by calculating the inverse of the slope of the linear regression line. Standard deviations of the pH and water-activity data were calculated.
Results Figs 1^4 show populations of L. monocytogenes during drying and storage of beef slices. Statistical analysis of the microbial data revealed that the main e¡ects of acid-adaptation, predrying treatment, drying time and agar media, and the interactions of acid adaptation predrying treatment drying time and predrying treatment drying time agar media were signi¢cant (Po0?05) for drying but not (PZ0?05) for storage data.
Temperature Changes in air temperature and meat surface temperatures during drying are shown in Fig. 5. During loading of the preheated dehydrator trays with the meat slices, the air temperature decreased from 601C to 401C at the beginning of drying, and then gradually increased back to 60711C within approximately 4 h. Surface temperature of meat slices from all trays reached 60721C within approximately 6 h. At the end of drying, surface temperatures of meat slices decreased to room temperature (c. 25^301C) within approximately 1 h.
E¡ect of agar media Signi¢cant di¡erences (Po0?05) in numbers of bacteria recovered on TSAP and PALCAM within each treatment were observed after 4 and 7 h of drying in MM, AATM and TWTM when the product was inoculated with non-adapted cultures. In products inoculated with acid-adapted cultures, signi¢cant di¡erences were observed in MM only after 4 h of drying. Bacterial counts recovered on two agar media from C and TM products were not significantly (PZ0?05) di¡erent during drying, but on day 15 during storage.These results indicate that MM, AATM and TWTM treatments
550 M. Calicioglu et al.
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+ Traditonal marinade (TWTM)
7 6
r2 0.99 0.96 0.93 0.95 0.99
D-value (h) 2.17 2.22 1.80 1.76 1.54
Log10 CFU/cm
2
5 4 3 2 1 0 -1 AI
0
2
4
6
8
10
Time (h)
(A)
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7
D-value (h) 2.11 2.15 1.66 1.52 1.51
2
r 0.99 0.96 0.90 0.91 0.88
6
Log10 CFU/cm 2
5 4 3 2 1 0 -1 AI
(B)
0
2
4
6
8
10
Time (h)
Figure 1. Survival of non-acid adapted L. monocytogenes as determined on TSAP (A) and PALCAM (B) during preparation (marination at 41C, 24 h) and drying (601C, 10 h) of beef jerky treated with various marinades (n = 4). AI: after inoculation; 0: after marination.Vertical bars are the standard deviation of the mean.
caused signi¢cant cellular injury to L. monocytogenes during drying while C and TM treatments resulted in only limited injury to cells.
E¡ect of predrying treatments Initial numbers of bacteria were signi¢cantly (Po0?05) reduced (1?0^1?7 log) after applica-
Jerky processing to inactivate Listeria 551
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7
D-value (h) 1.84 2.14 1.24 1.27 1.28
r2 0.90 0.94 0.91 0.84 0.87
6
Log10 CF U/cm
2
5 4 3 2 1 0 -1 AI
0
2
4
6
8
10
Time (h)
(A)
D- value (h) 1.90 2.10 1.20 1.20 1.27
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
7 6
r2 0.92 0.98 0.80 0.81 0.81
Log10 CF U/ cm
2
5 4 3 2 1 0 -1 AI
(B)
0
2
4
6
8
10
Time (h)
Figure 2. Survival of acid-adapted L. monocytogenes as determined onTSAP (A) and PALCAM (B) during preparation (marination at 41C, 24 h) and drying (601C, 10 h) of beef jerky treated with various marinades (n = 4). AI: after inoculation; 0: after marination.Vertical bars are the standard deviation of the mean.
tion of AATM and TWTM and holding meat slices at 41C for 24 h, irrespective of acid-adaptation or agar media. Application of MM significantly reduced the populations of bacteria in products inoculated with non-adapted culture
only (Figs 1 and 2). Reductions ( 0?2 to 0?7 logs) in C and TM were not signi¢cant, irrespective of acid-adaptation. These results may indicate that immersing inoculated meat slices with L. monocytogenes in 5% acetic acid solution
552 M. Calicioglu et al.
3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditonal marinade (TWTM)
Log10 CFU/cm2
2
1
0
-1 0
10
20
30
40
50
60
Day
(A)
3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1 0
10
20
(B)
30
40
50
60
Day
Figure 3. Fate of surviving non-acid-adapted L. monocytogenes during aerobic storage at 251C for 60 days on beef jerky after treating with various marinades and drying at 601C for 10 h, as determined on TSAP (A) and PALCAM (B) (n = 4). 0 d: Bacterial counts at 10 h are included as day 0 counts for statistical analysis of the storage data.Vertical bars are the standard deviation of the mean.
followed by storage at 41C for 24 h are detrimental to L. monocytogenes, and potential cell detachment caused by Tween 20 was minimal or did not assist in increasing the subsequent killing e¡ect of acetic acid.
E¡ect of drying Irrespective of acid-adaptation or the recovery media used, major reductions (Po0?05) in bacterial counts occurred in all treatments
Jerky processing to inactivate Listeria 553
3 Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1
0
10
20
30
40
50
60
Day
(A) 3
Control (C) Traditional marinade (TM) Modified marinade (MM) Acetic acid+Traditional marinade (AATM) TW20+Acetic acid+Traditional marinade (TWTM)
Log10 CFU/cm 2
2
1
0
-1 0
(B)
10
20
30
40
50
60
Day
Figure 4. Fate of surviving acid-adapted L. monocytogenes during aerobic storage at 251C for 60 days on beef jerky after treating with various marinades and drying at 601C for 10 h, as determined on TSAP (A) and PALCAM (B) (n = 4). 0 d: Bacterial counts at 10 h are included as day 0 counts for statistical analysis of the storage data.Vertical bars are the standard deviation of the mean.
during the ¢rst 4 h of drying, while declines between 4 and 7 h of drying were smaller (Figs 1 and 2). No signi¢cant reduction in bacterial populations was found in any treatment inoculated with either acid- or non-
adapted cultures between 7 and 10 h of drying. In general, drying of MM, AATM, and TWTM products inoculated with both culture types resulted in signi¢cantly lower bacterial populations, as determined with both TSAP and
554 M. Calicioglu et al.
70
Temperature (°C)
60
50
40
30
Dehydrator air temperature Beef slice surface (bottom tray) Beef slice surface (middle tray) Beef slice surface (top tray)
20
10 0
2
4
6
8
10
12
Drying time (h)
Figure 5. Mean (n = 4) temperatures of dehydrator air (middle hole air temperature) and the surface of beef slices during drying at 601C for 10 h using a home-type food dehydrator.
PALCAM, than did drying of C and TM treatments. There was no signi¢cant (PZ0?05) di¡erence in inactivation of acid-vs non-adapted inocula within individual treatments. However, numbers from AATM and TWTM treatments dropped below the detection limit (o 0?4 log cfu cm 2 ) of direct plating in products inoculated with acid-adapted cultures by 4 h of drying, whereas bacteria were still countable in products inoculated with non-adapted culture at 10 -h drying, indicating that acid-adapted cells may have been slightly more sensitive to these treatments. Bacterial counts in the C and TM treatments were not signi¢cantly different during drying, irrespective of agar media or acid-adaptation. Total log reductions in the numbers of bacteria in the products inoculated with nonadapted L. monocytogenes at the end of 10 h of drying as determined on non-selective (TSAP) and selective (PALCAM) media, were 3?8^4?6 for C, 4?0^4?3 for TM, 5?6^5?9 for MM, 5?9^6?2 for AATM and 5?9^6?2 for TWTM. Correspond-
ing reductions in log counts of bacteria on products inoculated with acid-adapted cells were 4?0^4?2 for C, 4?0^4?3 for TM, 5?7^6?6 for MM, 5?7^6?7 for AATM, and 6?3^6?7 for TWTM. D-values calculated based on bacterial counts determined on each of the two agar media between 0 and 7 h during drying are shown in Figs 1 and 2. Irrespective of inoculum acid-adaptation, MM, AATM and TWTM treatments resulted in smaller D-values than the control and TM indicating faster declines in the numbers of bacteria.
E¡ect of storage Populations of surviving bacteria in all treatments continued to decrease especially during the ¢rst 10^30 days of the 60 -day storage at ambient temperature (251C) (Figs 3 and 4).The signi¢cant declines observed only in treatments C and TM between 0 (after drying) and 15 days, resulted probably from the fact that high numbers of bacteria survived drying in these two treatments. In general, bacterial counts on
Jerky processing to inactivate Listeria 555
MM, AATM and TWTM products dropped below the detection limit earlier than those on C and TM products probably because of the lower bacterial counts on day 0 in these treatments in combination with lower pH and Aw . No signi¢cant di¡erence in counts between agar media was observed in any of the treatments during the entire storage period; this indicates that death of bacteria was occurring in all treatments during the entire storage period. The earliest complete elimination of the L. monocytogenes (enrichment negative) during product storage occurred on day 15 in AATMand TWTM-treated jerky, regardless of acidadaptation and in MM when the product was inoculated with non-adapted culture. By day 60, bacterial counts in all treatments were below the detection limit by direct plating on PALCAM and also on TSAP, with the exception of C when inoculated with non-adapted culture. At the end of storage, viable cells were recovered from C only, regardless of acidadaptation.
pH and Aw Overall, application of MM, AATM and TWTM reduced the pH of meat compared to that of the C and TM product (Table 1). No appreciable change in pH of the products was observed during drying or storage. Water activity of products decreased remarkably, as expected, during drying (Table 2). However, no notable di¡erence was found among treatments during storage. The ultimate Aw of the ¢nished products varied between 0?600 and 0?700. Slight £uctuations between sampling points were probably because of variation among slices.
Discussion The present study revealed that inactivation of L. monocytogenes during drying of beef jerky was a¡ected greatly by the type of predrying treatment. Although drying of jerky in all treatments reduced bacterial populations, the
Table 1. Mean (n = 4; standard deviation) pH values of beef jerky slices inoculated with acid- or nonadapted Listeria monocytogenes and subjected to various predrying marination treatments before drying at 601C for 10 h and storage at 251C for 60 days Inoculated with acid-adapted L. monocytogenes Steps Processing
Time
Ca
TMb
MMc
0h
5?43 (0?13) 5?62 (0?05) 5?56 (0?02) 5?63 (0?05)
5?25 (0?11) 5?41 (0?14) 5?58 (0?03) 5?58 (0?05)
4?44 (0?20) 4?73 (0?03) 4?70 (0?04) 4?84 (0?06)
4?34 (0?12) 4?63 (0?08) 4?59 (0?08) 4?66 (0?03)
5?65 5?61 4?82 (0?02) (0?03) (0?09) Day 30 5?63 5?46 4?71 (0?05) (0?09) (0?24) Day 60 5?66 5?54 4?75 (0?11) (0?16) (0?15)
4?69 (0?04) 4?52 (0?13) 4?73 (0?13)
4h 7h 10 h Storage
a
Day 15
AATMd TWTMe
Inoculated with non-acid adapted L. monocytogenes C
TM
MM
AATM
TWTM
4?29 (0?12) 4?58 (0?11) 4?58 (0?04) 4?65 (0?01)
5?69 (0?03) 5?78 (0?04) 5?74 (0?12) 5?70 (0?06)
5?57 (0?02) 5?75 (0?08) 5?68 (0?12) 5?62 (0?09)
4?61 (0?07) 4?77 (0?09) 4?77 (0?15) 4?83 (0?06)
4?62 (0?08) 4?84 (0?07) 4?91 (0?03) 4?88 (0?12)
4?61 (0?11) 4?85 (0?03) 4?80 (0?04) 4?72 (0?11)
4?72 (0?04) 4?63 (0?11) 4?75 (0?11)
5?48 (0?14) 5?41 (0?05) 5?55 (0?06)
5?39 (0?16) 5?35 (0?11) 5?53 (0?09)
4?54 (0?32) 4?55 (0?04) 4?76 (0?07)
4?51 (0?18) 4?61 (0?18) 4?72 (0?18)
4?40 (0?24) 4?61 (0?14) 4?69 (0?16)
No predrying treatment or marinade prior to refrigeration of inoculated slices at 41C for 24 h and drying. Marinated with TM (pH 4?3), held at 41C for 24 h, and then dried. c Marinated with modi¢ed marinade (1?2% lactate, 9% acetic acid, and soy sauce w/ 5% ethanol) ( pH. 3?0), held at 41C for 24 h, and then dried. d Dipped into 5% acetic acid solution (pH 2?5) for 10 min at ambient temperature, drained for 2 min, and then marinated with TM. e Dipped into 1% Tween 20 (pH 6?6) for 15 min, and then 5% acetic acid solution for 10 min at ambient temperature, followed by marination with TM. b
556 M. Calicioglu et al.
Table 2. Mean (n = 4; standard deviation) Aw values of beef jerky slices inoculated with acid- or nonadapted L. monocytogenes and subjected to various predrying marination treatments before drying at 601C for 10 h and storage at 251C for 60 days Inoculated with acid-adapted L. monocytogenes Steps Processing
Time
Ca
TMb
MMc
0h
0?965 (0?005) 0?911 (0?018) 0?828 (0?010) 0?659 (0?016)
0?956 (0?003) 0?793 (0?085) 0?697 (0?107) 0?642 (0?023)
0?945 (0?001) 0?798 (0?023) 0?721 (0?006) 0?481 (0?044)
0?949 (0?006) 0?851 (0?074) 0?638 (0?033) 0?684 (0?034)
0?645 0?610 0?603 (0?009) (0?050) (0?011) Day 30 0?650 0?657 0?657 (0?007) (0?061) (0?002) Day 60 0?664 0?646 0?627 (0?014) (0?032) (0?021)
0?621 (0?049) 0?625 (0?003) 0?615 (0?021)
4h 7h 10 h Storage
Day 15
AATMd TWTMe
Inoculated with non-acid adapted L. monocytogenes C
TM
MM
AATM TWTM
0?950 (0?010) 0?878 (0?031) 0?786 (0?096) 0?579 (0?097)
0?961 (0?002) 0?900 (0?008) 0?747 (0?023) 0?697 (0?081)
0?948 (0?004) 0?904 (0?018) 0?804 (0?095) 0?642 (0?064)
0?955 (0?002) 0?830 (0?013) 0?603 (0?087) 0?567 (0?027)
0?623 (0?018) 0?684 (0?002) 0?643 (0?004)
0?667 0?633 0?597 0?613 0?668 (0?016) (0?027) (0?083) (0?021) (0?024) 0?652 0?624 0?606 0?643 0?662 (0?003) (0?001) (0?008) (0?061) (0?031) 0?666 0?602 0?575 0?636 0?658 (0?020) (0?066) (0?010) (0?065) (0?011)
0?950 (0?004) 0?793 (0?124) 0?690 (0?011) 0?658 (0?099)
0?952 (0?005) 0?889 (0?068) 0?774 (0?154) 0?618 (0?037)
a
No pre-drying treatment or marinade prior to refrigeration of inoculated slices at 41C for 24 h and drying. Marinated with TM (pH 4?3), held at 41C for 24 h, and then dried. c Marinated with modi¢ed marinade (1?2% lactate, 9% acetic acid, and soy sauce w/ 5% ethanol) (pH. 3?0), held at 41C for 24 h, and then dried. d Dipped into 5% acetic acid solution (pH 2?5) for 10 min at ambient temperature, drained for 2 min, and then marinated with TM. e Dipped into 1% Tween 20 (pH 6?6) for 15 min, and then 5% acetic acid solution for 10 min at ambient temperature, followed by marination with TM. b
modi¢ed marinades MM, AATM and TWTM applied as predrying treatments led to greater levels of reduction during drying. Survival of the pathogen during drying was not signi¢cantly di¡erent between control and traditionally marinated (TM) jerky, in the present study, indicating no additional antimicrobial e¡ect from TM. Although TM contains Worcestershire sauce, it was not acidic enough (pH 4?3) to cause an appreciable reduction in the pH of the TM product compared to control products, given the limited volume used per beef slice. The e¡ectiveness of TM in facilitating reduction in numbers of L. monocytogenes and other pathogens during drying was studied by other researchers. Harrison and Harrison (1996) reported that drying traditionally marinated whole muscle jerky at 601C for 10 h was su⁄cient to deliver 45?0 -log units reduction of E. coli O157:H7, L. monocytogenes, and S. typhimurium, as determined with selective agar media. In a more comprehensive study, Harrison et al. (2001) compared the e¡ectiveness of TM, oven-
heating at 711C before drying, boiling in marinade prior to drying, and post-drying heating of traditionally marinated jerky at 57?31C for 10 min in reducing numbers of E. coli O157:H7, L. monocytogenes, and Salmonella on whole muscle beef jerky during drying at 601C for 10 h. Their results showed that heating and boiling before drying reduced the numbers of all three pathogens, prior to drying, below the detection limit on selective agar media (MOX for Listeria), indicating an immediate action. At the end of drying, total reductions from all treatments were equal to or greater than 5?8, 3?9, and 4?6 -log units for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively, even with TM. Consumer acceptability of product heated after drying or boiled in marinade before drying was signi¢cantly higher than that for product heated before drying. The authors concluded that oven-heating jerky after drying resulted in safer and acceptable jerky product. In the present study, C and TM resulted in lower bacterial reductions
Jerky processing to inactivate Listeria 557
compared to use of MM, AATM or TWTM during drying, regardless of acid adaptation. In other words, at the end of drying, signi¢cantly higher numbers of bacteria were recovered from C and TM jerky on both TSAP and PALCAM than from jerky of other treatments. These results might indicate that predrying treatments MM, AATM and TWTM can improve the e¡ectiveness of drying on survival of L. monocytogenes further than TM alone. It has been reported that simultaneous exposure of bacteria to di¡erent stress factors requires increased energy consumption and leads bacteria to cellular death through metabolic exhaustion and disturbed homeostasis (Leistner 2000). Barker and Park (2001) reported that combinations of osmotic stress (NaCl, sucrose or glycerol), ethanol (5%), and high acid (pH 3?0, HCl) or 50 mmol concentrations of food-grade acid salts (including DL -lactate) provided reductions of stationary phase L. monocytogenes up to 5 logs in 4 min. Their results further indicated that such combination of organic acids, ethanol, and low pH killed the cells by damaging the bacterial cell membrane. This phenomenon was modi¢ed and adopted as a predrying treatment (modi¢ed marinade or MM) in the present study, by using commercially available soy sauce containing approximately 5% ethanol, and adding 2% of a 60% sodium lactate preparation, and 9% acetic acid to TM.The volume of marinade solution was also increased by using double the volume of soy sauce and Worcestershire sauce to deliver a higher marinade solution per slice than TM. The MM used in the present study was shown to be signi¢cantly more e¡ective in reducing the populations of L. monocytogenes cells on beef slices during drying compared to the use of TM and C, regardless of acid-adaptation. These ¢ndings indicated that combinations of lactate, ethanol and acetic acid might have a potential to be used in destruction of pathogenic bacteria in processed foods. It is known that bacterial cells attached to the surface of a product (such as meat) become more resistant to stress factors (such as heat) than non-attached cells (Humphrey et al. 1997). Another study showed that prespraying beef carcasses inoculated with high levels of E. coli 0157:H7 with 5% Tween 20 followed by
spraying with 2% lactic acid resulted in significantly higher reduction of the pathogen compared to spraying with lactic acid alone or water (Calicioglu et al. 2002). It has been speculated that Tween 20 may loosen cells or prevent cellular attachment on the meat surface via its surfactant and hydrophobic e¡ects, thus making the cells more vulnerable to the e¡ect of subsequent acid exposure. This proposed carcass decontamination method was applied as a predrying treatment in the present study (TWTM) prior to acetic acid dip and marinating with a TM recipe. Tween 20 (polysorbate 20, or polyoxyethylene-20 -sorbitan monolaurate) is permitted to be used in food as adjuvant (Code of Federal Regulations 2001). In the present study, there was no signi¢cant di¡erence in destruction of bacteria during drying between AATM and TWTM, regardless of acid adaptation, sampling time or recovery media. Both treatments were signi¢cantly more e¡ective than C and TM, even before drying. Lesser resistance of L. monocytogenes to acid conditions compared to E. coli O157:H7 (Roering et al. 1999, Samelis et al. 2001b) might explain this observation. In general, numbers of bacteria continued to decline in all treatments until 7 h of drying and then followed a much slower reduction rate, or did not change. This decelerated reduction in counts has been described as a ‘‘tailing e¡ect’’. In the present study, the tailing e¡ect was seen in treatments involving MM, AATM and TWTM, particularly, as determined by selective agar media, between 4 or 7 and 10 h of drying. Buchanan et al. (1994) investigated nonthermal inactivation of L. monocytogenes and reported that the tailing e¡ect was not because of the presence of a more resistant subpopulation. Their results revealed that inactivation rates of the pathogen in liquid media, as affected by pH and sublethal temperatures (up to 401C), were independent of initial population density. In contrast, as mentioned, preculturing of L. monocytogenes in acidic environment or in BHI broth with 0?2^0?5% glucose (acid-adaptation) increased the general acid resistance of the pathogen (Buchanan et al. 1994). There has been additional evidence that acid-adaptation of L. monocytogenes enhances its survival in acidic foods and that it may
558 M. Calicioglu et al.
increase cross-protection to other type of stresses (Gahan et al. 1996, Lou and Yousef 1997, Saklani-Jusforgues et al. 2000, Francis and O’Beirne 2001). In the present study, however, no enhanced survival of acid-adapted L. monocytogenes was observed during drying in any treatment. Similar results have been reported for other acid-adapted pathogens, such as Salmonella and Escherichia coli O157:H7, exposed subsequently to acid or additional stresses (e.g. heat, osmotic stress) during a food process (Dickson and Kunduru 1995, Ryu et al. 1999, Riordan et al. 2000). For example, Ryu et al. (1999) compared the survival of acidadapted, non-adapted and acid-shocked cells of E. coli O157:H7 in dried beef powder during storage.They reported that there was no significant di¡erence in survival of these cell types of the pathogen. The authors then concluded that acid-adaptation did not result in crossprotection against dehydration or osmotic stress. This di¡erence might be the result of use of multiple hurdles involved in the meatdrying process such as high acid, temperature, and low Aw as well as the in£uence of acidadaptation. It might be speculated that level of cross-protection from acid-adaptation is dependent on the presence and severity of other stress factors as a function of time. It seems, in the present study, that acid (e.g. rpH 4?8), heat (i.e.Z601C) and low water activity (o0?80) may provide a combination of hurdles able to overcome any resistance provided by acid-adaptation. In addition, the physiological state of the cells during treatment may in£uence their resistance or cross-protection behavior (McClements et al. 2001). In conclusion, results of the present study indicated that acid-adapted cells of L. monocytogenes did not exhibit any increased resistance to the drying process compared to that of nonadapted cells. Our results also revealed that using food-grade chemicals as predrying treatments (MM, AATM, and TWTM) improved the e¡ectiveness of the meat-drying process for inactivating L. monocytogenes, compared to the traditional jerky-making process. Studies of the e¡ects of such treatments on the sensory attributes of the ¢nal product are in progress. Under the conditions described in the present study, use of MM, AATM or TWTM in the
jerky-making process and storing the dried products for 15 days at ambient temperature, or use of TM and then storing for 60 days prior to consumption, should inactivate 6^7 logs of the pathogen.
Acknowledgements This project was funded by USDA-CSREES and by the Colorado Agricultural Experiment Station. We thank Excalibur Seasoning Company, and Heller Seasoning and Ingredients Inc., for providing irradiated spices.
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