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High-pressure processing and boiling water treatments for reducing Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus during beef jerky processing
Food Control 39 (2014) 105e110
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High-pressure processing and boiling water treatments for reducing Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus during beef jerky processing Joshua A. Scheinberg, Amanda L. Svoboda, Catherine N. Cutter* Department of Food Science, The Pennsylvania State University, University Park, 202 Food Science Building, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 31 October 2013 Accepted 1 November 2013
Beef jerky is a convenient, ready-to-eat meat product, but requires processing lethality steps to ensure the safety of the product. Previous outbreaks involving various jerky products have highlighted the risks associated with jerky and the importance of utilizing pathogen interventions during processing. In this study, two alternative interventions were evaluated for reducing pathogen populations during jerky processing. Results demonstrated that high pressure processing (HPP; two treatments of 550 MPa, 60 s) could produce significant (p < 0.05), but variable reductions (6.83 and 4.45 log10 CFU/strip) of Salmonella spp. and Escherichia coli O157:H7, respectively, on resulting beef jerky. HPP treatments, however, produced minor reductions (p < 0.05) of Gram-positive pathogens, resulting in reductions of 1.28 and 1.32 log10 CFU/strip of Listeria monocytogenes and Staphylococcus aureus, respectively. Alternatively, boiling water (100 2 C) treatments (20e30 s) used after marination and prior to dehydration, reduced Salmonella spp., E. coli O157:H7, L. monocytogenes, and S. aureus populations >5.0 log10 CFU/strip in resulting beef jerky. Thus, 20 or 30 s boiling water (100 2 C) treatments could be effective interventions for commercial jerky processors or home food preservers. Future validation of these processes in-plant could provide processors and regulators with alternative strategies for safe and shelfstable jerky products. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Jerky Pathogens High pressure processing Boiling water Intervention
1. Introduction Beef jerky and other dried meat snacks have continued to grow in popularity. In fact, these products are among the Top 10 Consumer Packaged Goods Categories, showing considerable growth in sales in the U.S. during the last decade (Roberts, 2003; Sloan, 2011). Although a few large beef jerky processors currently dominate 70% of U.S. jerky sales, small niche meat processors continue to make a wide range of unique jerky and dried meat snacks throughout the country (Schaefer, 2013). Commercially-processed, whole-muscle beef jerky is a convenient, ready-to-eat (RTE) meat product, but requires sufficient processing lethality steps to ensure the safety of the product throughout its shelf life. Previous outbreaks involving various jerky products have highlighted the risks associated with meat jerky and the importance of validating jerky processing methods. The most common pathogen associated with beef jerky outbreaks has been Salmonella, with the largest outbreak occurring in 1994, affecting 93 persons who consumed beef jerky made in
* Corresponding author. Tel.: þ1 814 865 8862; fax: þ1 814 863 6132. E-mail address: [email protected] (C.N. Cutter). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.11.002
New Mexico (CDC, 1995). A more recent outbreak in 2012 involved 4 cases of salmonellosis from individuals who consumed turkey jerky made in Minnesota (MDH, 2012). Additionally, consumption of venison jerky has resulted in infections with Escherichia coli O157:H7 in Oregon in 1994 and Connecticut in 2002 (Keene et al., 1997; Rabatsky-Her et al., 2002). In an effort to control spoilage and pathogenic microorganisms during jerky processing, many processors have sought alternative intervention methods. High pressure processing (HPP) is one such method that has seen a rise in its use among meat processors. HPP was first used on meat products by Macfarlane (1973) to increase the tenderness of meat; however its lethal effect on bacteria is considered an important reason to use this technology (Garriga & Aymerich, 2009; Simonin, Duranton, & De Lambaallerie, 2012). Cell death by HPP is achieved through a combination of effects, with cell membrane alteration as a primary cause of cellular death (Moussa, Perrier-Cornet, & Gervais, 2006; Patterson, 2005; Simonin et al., 2012). Using HPP, variable pathogen reductions have been demonstrated on several dried meat products. Jofré, Aymerich, Grébol, and Garriga (2009) demonstrated a >3.0 log unit reduction of Listeria monocytogenes and Salmonella spp. on dried cured ham when HPP was applied at 600 MPa for 6 min Porto-Fett et al.
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(2010) observed a >5 log unit reduction of E. coli O157:H7 on dried salami when treated with 600 MPa for up to 5 min. To our knowledge, no published study has examined the effects of HPP on beef jerky products experimentally inoculated with pathogens. Although generally regarded as an expensive alternative to other antimicrobial interventions, the use of HPP in the food industry has become more widespread in the last decade (Patterson, 2005). Custom HPP systems range between $500,000 and $2.5 million; however numerous facilities across the U.S. now offer HPP services (Balasubramaniam, Farkas, & Turek, 2008; Patterson, 2005). Although HPP service costs will vary due to different operating parameters, product specifications, packaging, volume, and labor costs, available sources have estimated treatment costs ranging between $0.04 and $0.11 per pound of product (Balasubramaniam et al., 2008; Patterson, 2005). Depending on the resources and product specifications, HPP may be a useful and feasible antimicrobial intervention for the jerky processor. Nevertheless, many jerky processors have sought other alternative processes and focused on improvements to marinade recipes or altered heat treatments, rather than utilize processes like HPP. One such strategy is to heat raw meat strips in a boiling marinade prior to dehydration. Harrison, Harrison, Rose-Morrow, and Shewfelt (2001) demonstrated >5 log unit reduction of E. coli O157:H7, L. monocytogenes, and Salmonella after beef strips were marinated and boiled for 5 min in the marinade, prior to dehydration. However, the 5 min boiling method produced significant changes to the color and saltiness of the finished jerky compared to jerky that had only been marinated and dehydrated. It is unknown whether boiling treatments of less than 5 min prior to dehydration would be as effective in reducing pathogens. It is also unknown whether variations in marinade recipes would alter the effectiveness of this boiling marinade technique, and whether they may contribute to the sensory changes. The use of boiling water and minimum boiling water exposure times (<1 min), rather than a boiling marinade for several minutes, may offer a more suitable and consistent alternative for jerky processors seeking additional lethality processes that produce minimal sensory changes to the final jerky product. Jerky processed and sold commercially is regulated by the USDA-Food Safety and Inspection Service (USDA-FSIS). Currently, the USDA-FSIS Jerky Compliance Guidelines provide recommended time, temperature, and relative humidity (RH) processing parameters to achieve necessary reductions in selected pathogens for jerky processing (USDA-FSIS, 2012a). The USDAFSIS recommends that current jerky processing methods should achieve 5 log unit reductions of Salmonella spp., E. coli O157:H7, and at least a 3 log unit reduction of L. monocytogenes in the final jerky product (USDA-FSIS, 2012a). Many small and very small processors may choose to produce jerky products that may not follow typical processing parameters as recommended by the USDA-FSIS. In these instances, scientific support must be provided to demonstrate that the processes used produce the necessary reductions of Salmonella spp., E. coli O157:H7, and L. monocytogenes in the final jerky product. Due to the increasing popularity of home jerky processing and the production of unique jerky products by processors who may not utilize typical dehydration times and temperatures, as recommended by the USDA-FSIS, the purpose of this study is to investigate the efficacy of two alternative antimicrobial interventions (HPP and submersion in boiling water (100 2 C)), for use in applications where standard heat and drying lethality treatments for jerky processing may not be implemented. The parameters used in this study mimic those selected by a very small jerky processor seeking additional antimicrobial interventions for their nontraditional jerky product.
2. Materials and methods 2.1. Preparation of bacterial inoculum Cultures of Salmonella Typhimurium ATCC 14028, Salmonella Enteritidis ATCC 13076, E. coli O157:H7 ATCC 43889, L. monocytogenes Scott A, and a human Staphylococcus aureus isolate obtained from the Food Microbiology Culture Collection at The Pennsylvania State University Food Science Department were used to produce the inoculum cocktail. All cultures were stored at 80 C in tryptic soy broth (TSB; Becton Dickinson and Company; BD, Sparks, MD) supplemented with 20% (v/v) glycerol to ensure viability. Pathogen cultures were prepared by transferring the frozen stock to fresh TSB and incubating at 37 C for 24 h. Colonies were later isolated by streaking TSB cultures to tryptic soy agar (TSA; BD). Single colonies of each pathogen were isolated and transferred to fresh TSB and repeated sequentially in triplicate to ensure pure culture. Isolated colonies of each pathogen were confirmed using Gram stains and Salmonella spp., L. monocytogenes, Staphylococcus aureus, and E. coli O157:H7 agglutination kits (Remel, Lenexa, KS; Oxoid, Basingstoke, Hampshire) prior to inoculum preparation. Inoculum was prepared using the methodologies described by Richard and Cutter (2011), in which single colonies of each pathogen were transferred to 500 mL of TSB, in triplicate, and grown statically at 37 C for 24 h to obtain cell concentrations of 9 log10 CFU/ml. As was also described in Richard and Cutter (2011), equal volumes of each pathogen culture (1500 ml each) were mixed in a large sterilized metal bin to produce a non-diluted cocktail inoculation bath for the raw beef strip inoculation. This inoculum preparation allowed for the simultaneous inoculation of large quantities of beef samples. 2.2. Preparation and inoculation of raw beef strips Beef top rounds were purchased from a local supermarket and transported in an insulated cooler with ice to the Penn State Muscle Foods Microbiology Laboratory and stored at 20 C until further use. Frozen beef was thawed for approximately 24 h at 4 C to soften the meat, aseptically removed from the packaging, and all visible fat was removed with a sterile knife. Beef strips were prepared using a sanitized mechanical meat slicer (Globe; Dayton, OH) producing 3.2 mm thick, uniform pieces. Each beef strip was trimmed to 10.2 cm 5.0 cm, packaged in sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI), and stored at 4 C until fully defrosted. For both the HPP and boiling water submersion experiments, prepared beef strips (n ¼ 70 for HPP treatment; n ¼ 57 for boiling water treatment) were placed aseptically into the inoculum bath for 30 min under a biological safety hood with frequent stirring and agitation. Following inoculation, each beef strip was removed from the inoculum aseptically using sterile forceps, allowed to drip for 3e5 s, and placed onto sterile drip trays. Beef strips were allowed to dry on the drip trays under the biological safety hood for 15 min prior to each jerky experiment. After air-drying, beef strips were placed into a bowl of pre-prepared marinade solution, mixed well, covered with plastic wrap, and allowed to marinate for 24 h at 4 C. Following marination, beef strips were removed from the marinade, allowed to drip for 3e5 s, and placed onto sterile drip trays under the biological safety hood for 15 min prior to further treatments. The marinade solution was prepared by mixing 57 g of a proprietary spice blend (Lazy Jims Jerky Spice; York Springs, PA) with 237 ml of sterile water for every 454 g (1 pound) of beef. This solution was heated to 83 C and cooled to 25 C prior to use, in accordance with the recipe and methods provided by the manufacturer.
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2.3. Boiling water submersion treatments Inoculated and marinated raw beef strips used for boiling water submersion treatments (n ¼ 57) were aseptically transferred to a sterilized, metal wire basket (n ¼ 9 per basket/time point), covered with foil, and stored aseptically at 4 C prior to the boiling water treatment. Four liters of sterile water was prepared for each of the boiling water exposure time trials (0, 5, 10, 15, 20, 30 s). In each trial, 4 L of sterile water was poured into a sterile metal bin sitting on two pre-heated laboratory hot plates (25.4 cm 25.4 cm; VWR, NY), covered with foil, and heated until boiling (100 2 C). Each metal basket (one per time point) containing the inoculated and marinated beef strips was lowered into the boiling water (100 2 C), ensuring all strips were submerged, and held for the respective time. Since the temperature of the water typically dropped below boiling after the addition of the beef strips, the exposure time was measured once boiling resumed, 15 s. Following the boiling treatment, baskets were removed from the water and beef strips aseptically transferred onto dehydrator drying racks, and allowed to dry in the biological safety hood for 15 min. Boiled beef strips were then dehydrated using a 9-tray dehydrator (Excalibur Dehydrators, Fort Lauderdale, FL) and dried at a temperature of 55 C for 4 h which was contained in a biological safety hood with drying racks rotated each hour. The temperature of the dehydrator was verified to be at 55 C prior to the dehydration process to account for dehydrator come up time. Temperatures were monitored every 30 min using a thermometer (VWR) placed inside the dehydrator, and taken at several locations within the dehydrator to ensure beef slices were exposed to the same temperature. Relative humidity (RH) measurements were also taken every hour, although the Excalibur Dehydrator is not designed to control RH. Temperatures fluctuated in certain areas by 1e5 C. Internal jerky temperatures were verified to have reached 55 C following the 4-h dehydration cycle. After dehydration, beef jerky slices were placed into vacuum packaging bags (15.2 cm 20.3 cm; Prime Source, Kansas City, MO) and vacuum sealed (3 pieces of jerky/bag) using an Ultravac UV-250 vacuum sealer (UltraSource USA, Kansas City, MO). Raw beef strips and jerky subjected to boiling water (100 2 C) submersion treatments were sampled for microbiological analysis following each of the steps (n ¼ 3 beef strips or jerky per processing step and time point): inoculation, marination, dehydration, 1 h following vacuum packaging, and after 7 and 14 days of vacuum packagedstorage at room temperature (22e25 C).
transferred to a filtered stomacher bag (Interscience, St. Rockland, MA), weighed, and diluted with 10 times the volume of the sample with Buffered Peptone Water (BPW; BD) and stomached for 2 min at 230 rpm (Stomacher 400; Seward, West Sussex, UK). The stomachate was serially diluted in BPW and aliquots of 0.1 ml and 0.5 ml were spread plated in duplicate and quadruplicate, respectively, onto selective agars to achieve a detection limit of 1 log10 CFU/strip. Selective agars used for the enumeration of surviving pathogens included: Xylose Lysine Deoxycholate agar (XLD; BD), Baird-Parker agar with egg yolk tellurite (Remel), Cefixime Tellurite-Sorbitol MacConkey agar (CT-SMAC; Remel), and Modified Oxford agar with antibiotic supplement (MOX; Remel) for enumeration of Salmonella spp., S. aureus, E. coli O157:H7, and L. monocytogenes populations, respectively. Samples also were transferred to enrichment media to determine the presence or absence of surviving cells in those samples with low pathogen concentrations. Pathogens were enriched using methods adopted from the USDA-FSIS Microbiological Laboratory Guidebook (MLG; USDA-FSIS, 2012a, 2012b). Briefly, enrichment of Salmonella spp. utilized a non-selective preenrichment of lactose broth (BD), followed by selective enrichments of tetrathionate broth (BD) and selenite cystine broth (BD) with subsequent plating on XLD. UVM (BD) and Fraser broths (BD) were used as primary and secondary enrichments, respectively, for L. monocytogenes. Following the secondary enrichment step, samples were plated on MOX (BD). Gram-negative broth (GN; BD) and TSB w/5% NaCl (TSB; BD) were used as primary enrichments for E. coli O157:H7 and S. aureus, with subsequent plating on CT-SMAC and Baird-Parker agar, respectively. Measurements of pH and water activity (Aw) of all jerky samples were determined at each sampling time using a VWR Symphony pH meter (VWR) and Aqua Lab 4TE (Decagon Devices, Pullman, WA) water activity meter. 2.6. Statistical analysis All bacterial populations were converted to log10 CFU/strip for analysis and transformation of data was performed as necessary to meet the assumptions of the statistical test. Analyses of statistical tests were performed using SAS (SAS software, Version 9.3, SAS Institute Inc., Cary, NC). In cases where there was a zero count on duplicate plates, a count of 1 CFU was assigned to allow for count conversion to log10 CFU/strip. Comparisons of pathogen populations were determined using ANOVA with Tukey’s HSD test at a ¼ 0.05.
2.4. HPP treatment
3. Results
Inoculated and marinated raw beef strips used for HPP treatments (n ¼ 70) were immediately transferred to dehydrator drying racks, dehydrated using a 9-tray dehydrator, and vacuum packaged, as described in section 2.3. HPP was performed using the vertically loaded QFP 2L-700 High Pressure Laboratory Food Processing System (Avure, Franklin, TN) capable of operating at 100,000 PSI (689 MPa). Each vacuum packaged bag of beef jerky samples were high pressure-treated at 550 MPa for two consecutive, 60 s treatments at a process water temperature of 22 C. Raw beef strips and jerky used for the HPP treatments were sampled for microbiological analysis following each of the steps (n ¼ 10 beef strips or jerky per each processing step): inoculation, marination, dehydration, 1 h following vacuum packaging, HPP, and 7 and 14 days following vacuum-packaged storage at room temperature.
3.1. Boiling water submersion treatments
2.5. Microbiological analyses For both the HPP and boiling water submersion experiments, each individual raw beef strip and jerky were aseptically
107
Boiling water (100 2 C) treatments produced variable reductions in pathogen populations on beef slices (Figs. 1e6). Interestingly, populations of Salmonella spp., exposed to any of the boiling water treatments and subsequent dehydration, were reduced >5 log10 CFU/strip from pre-marination levels. Conversely, E. coli O157:H7, L. monocytogenes, and S. aureus populations were not reduced >5 log10 CFU/strip until after exposure to 20 and 30 s of boiling water and dehydration. As a control, beef slices not exposed to the boiling water treatment (0 s) were briefly immersed in water (20e22 C) to observe the isolated effects of the other processes (marinating, dehydration, and vacuum packaging). Control samples demonstrated reductions (<3 log10 CFU/strip) in E. coli O157:H7, L. monocytogenes, and S. aureus populations. Salmonella spp. populations were reduced 5 log10 CFU/strip after marination, dehydration, vacuum-packaging, and 24 h of vacuum-packaged storage. Boiling water treatments of 20 and 30 s resulted in log reductions >5 log10 CFU/strip after vacuum-packaging for all pathogens tested
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9
9
8
8
7
7
6 5 4
EC
6
EC
SS
5
SS
SA
3
LM
2
4
SA
3
LM
2 1
1
0
0
Inoculation
Boil
Dehydration
Inoculation
Vacuum
Fig. 1. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (0 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
Boil
Dehydration
Vacuum
Fig. 4. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (15 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
9 8 7 6
EC
5
SS
4
SA
3
LM
2 1 0
Inoculation
Boil
Dehydration
Vacuum
in this study, with Salmonella spp. reduced to levels below the detection limit and also negative for the pathogen following enrichment. Minimal changes in pH were observed among all treatments throughout the experiment; with a slight increase following marination and a final pH of 6.54 among 30 s treatment samples (Table 1). RH measurements taken during dehydration ranged from 10.4 to 52.7% on the low and high ends. Interestingly, initial aw levels of 0.98 among inoculated samples were reduced to 0.57 after 0 s treatments and to 0.64 among 30 s treatments, exhibiting less aw reduction as boiling water treatment time increased. These differences observed between final aw levels between each
9 8 7 6
EC
5
SS
4
SA LM
2
EC SS SA LM
* Inoculation
Fig. 2. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (5 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
3
9 8 7 6 5 4 3 2 1 0
1 0
Boil
Dehydration
Dehydration
Vacuum
Fig. 5. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (20 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation. * Pathogen populations were reduced below the detection limit and were negative for enrichment.
treatment, could suggest that longer boiling water exposures may produce a greater dilution of solutes present on marinated beef strips. 3.2. HPP treatments As expected, marination, dehydration, and vacuum packaging alone, did not result in >5 log10 CFU/strip reductions of the pathogens evaluated in this study (Table 2). Although reduction in aw occurred following dehydration (from aw of 0.97 to 0.74), these
9 8 7 6 5 4 3 2 1 0
EC SS SA LM
* Inoculation
Inoculation
Boil
*
Boil
Dehydration
* Vacuum
Vacuum
Fig. 3. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (10 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
Fig. 6. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (30 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation. * Pathogen populations were reduced below the detection limit and were negative for enrichment.
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Table 1 pH and aw values of beef samples during jerky processing and boiling water treatments.a Boiling water exposure times
Inoculation
Marinationb
Boiling water Treatment
0s
5.60 0.07 0.982 0.02 e
6.60 0.06 0.971 0.03 e
10 sc
e
e
c
e
e
20 sc
e
e
30 sc
e
e
6.90 0.957 6.93 0.954 6.86 0.951 6.89 0.940 6.70 0.943 6.63 0.954
5 sc
15 s
Dehydration
0.02 0.003 0.13 0.006 0.01 0.007 0.01 0.007 0.07 0.003 0.02 0.002
6.54 0.642 6.64 0.619 6.67 0.601 6.70 0.568 6.67 0.642 6.71 0.628
0.07 0.02 0.06 0.03 0.01 0.01 0.06 0.03 0.08 0.01 0.03 0.01
Vacuum package 6.54 0.577 6.67 0.581 6.57 0.616 6.52 0.611 6.68 0.629 6.54 0.649
0.07 0.04 0.04 0.02 0.07 0.01 0.07 0.02 0.06 0.006 0.08 0.008
Note: Analysis of pH and aw was performed immediately following each of the experimental steps (Inoculation, Marination, Boiling Water Treatment, Dehydration, 1 Hour Following Vacuum Packaging). a pH and aw values are mean of (n ¼ 3) for each processing step, pH values are listed on top of aw values for each processing step and exposure time. b pH of marinade solution was 6.70. c All beef strips (n ¼ 57) were inoculated and marinated together in the same containers, pH and aw was performed (n ¼ 3) following inoculation and marination which represented conditions of all beef strips prior to being separated into groups based on boiling water exposure times. Values listed for inoculation and marination at (0 s) are the same for the other exposure times, represented by a ().
jerky sensory characteristics (Harrison et al., 2001). In this study, boiling water (100 2 C) treatments of 30 s were selected to evaluate the efficacy of a rapid and easy method for pathogen reduction that could be employed by both commercial jerky processors or by the home food preserver. Our results demonstrate that the application of a 20e30 s boiling water submersion followed by dehydration and vacuum packaging can reduce pathogen populations (Salmonella spp., E. coli O157:H7, L. monocytogenes, and S. aureus) > 5 log10 CFU/strip. As such, this step may be suitable for meeting the lethality target recommended by current USDA-FSIS jerky processing guidance. It should additionally be noted that jerky exposed to boiling water treatments exhibited a final aw of 0.57e0.65 and a pH of 6.52e6.68 depending on the boiling water treatment time, which may be lower or higher than generally desired by certain processors. Current USDA-FSIS guidance recommends jerky be processed to achieve a final aw of 0.85 if packaged in an aerobic environment, although a critical limit of 0.91 could be acceptable if vacuum packaged (USDA-FSIS, 2012a). Current USDA-FSIS guidance does not provide specific pH values to be achieved in marinades or final jerky products, although it is recognized that pH is a critical parameter that can provide additional product safety but will vary depending on the process. The efficacy of HPP to reduce pathogens in food products has been studied extensively. However, little to no data exists on the effects of HPP for reducing bacterial pathogens associated with beef jerky. Due to the increase in HPP facilities around the country, processors of dried foods like beef jerky, could utilize HPP as an alternative lethality process to minimize the heat and dehydration treatments performed during commercial jerky processing. To date,
changes did not appear to drastically reduce pathogen populations; however reductions were statistically significant (p < 0.05). RH measurements taken during dehydration ranged from 8.2 to 51.5% on the low and high ends. HPP treatments of two cycles of 550 MPa for 60 s, were performed immediately following vacuum packaging of the dehydrated jerky, resulting in variable reductions in pathogen populations. The 2-cycle HPP treatment was found to have the greatest effect on Salmonella spp. populations with reductions of 3.2 log10 CFU/strip. It is important to note that reductions on individual samples were variable. Small reductions of L. monocytogenes (1.2 log10 CFU/strip) and S. aureus (1.3 log10 CFU/ strip) populations also were observed following HPP treatments; however, E. coli O157:H7 populations did not significantly change as an immediate result of HPP. Fourteen day storage (20e22 C) of HPP-processed samples resulted in further reductions in Salmonella spp. (1.4 log10 CFU/strip), and E. coli O157:H7 (1 log10 CFU/strip) populations. Populations of L. monocytogenes and S. aureus increased slightly following 14 day vacuum packaged storage; however these increases were likely due to higher variation among jerky samples. Interestingly, total log reductions of >5 log CFU/strip were observed in Salmonella spp. over the course of the experiment, with E. coli O157:H7 reductions reaching 4.4 log10 CFU/strip after 14 days of vacuum packaged storage. 4. Discussion Previous research utilizing applications of a heat treatment, prior to dehydration, has demonstrated variable effects on the reduction of pathogens, while also producing significant changes in
Table 2 Average counts (log10 CFU/strip) of L. monocytogenes, Salmonella spp., E. coli O157:H7, and S. aureus; pH and aw during jerky processing and subjected to HPP. Inoculation L. monocytogenes Salmonella spp. E. coli O157:H7 S. aureus pHx a wx
7.85 7.43 7.30 7.25 5.60 0.982
0.14a 0.10a 0.18a 0.14a 0.08 0.003
Marinationz 7.61 7.15 6.48 7.15 6.68 0.971
0.25a 0.19a 0.22b 0.40ab 0.07 0.003
Dehydration 6.85 5.57 6.12 6.66 6.71 0.735
0.17bc 0.40b 0.98b 0.37bc 0.02 0.05
Vacuum package 6.71 5.33 4.21 6.16 6.90 0.784
0.19c 0.33b 0.95c 0.39ce 0.03 0.07
HPP 5.51 2.07 3.82 4.83 6.77 0.751
HPP (7d)
0.19d 1.28c 0.89c 0.37d 0.04 0.02
6.49 1.60 3.41 6.01 6.82 0.78
0.63ce 0.96c 0.6d 0.5e 0.09 0.05
HPP (14d)
Log10 CFU/strip Reductiony
6.57 0.47bce 0.6 0.57d 2.85 0.49e 5.93 0.29c 6.87 0.03 0.61 0.02
1.28 6.83 4.45 1.32
Note: Log10 CFU/strip counts and standard deviations are mean of (n ¼ 10; individual beef strips or jerky) for each processing step. Microbiological and pH and aw analysis was performed immediately following each of the experimental steps (Inoculation, Marination, Dehydration, 1 h Following Vacuum Packaging, HPP, HPP (7 d), HPP (14 d)). aee Different letters represent significant difference between processing steps within each pathogen (p < 0.05). x pH and aw values are mean of (n ¼ 3) for each processing step. y Log10 CFU/strip reduction was calculated by subtracting populations subjected to HPP (14 d) from initial, untreated inoculation counts. z pH of marinade solution was 6.70.
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research on HPP treatments of dried meat products has demonstrated bacterial pathogen reductions when utilizing pressures >550 MPa for >5 min. At these pressures and times, significant changes in meat color and water content also have been documented (Ferrini, Composada, Aranu, & Gou, 2012). In the current study, minimal HPP exposures (2 treatments of 60 s) were used in an effort to minimize changes in sensory characteristics (color, texture, etc.) of the beef jerky, although those effects were not investigated. The results of this study demonstrated significant reductions (p < 0.05) in all pathogens exposed to the HPP treatments. However, only Salmonella spp. populations were reduced enough to satisfy USDA-FSIS regulatory requirements. E. coli O157:H7 levels were not reduced by 5 log10 CFU/strip, although HPP treatment did significantly affect the pathogen, resulting in 4.4 log10 CFU/strip reductions over the 14 day experiment. Alternatively, HPP was less effective against the Gram-positive pathogens, since S. aureus and L. monocytogenes were minimally reduced by 1.0 log10 CFU/strip at day 14. Garriga, Aymerich, Costa, Monfort, and Hugas (2002) observed similar results, reporting recovery (3 log units) of L. monocytogenes and S. aureus during up to 20 day storage of inoculated, cooked ham slurry exposed to 400 MPa for 10 min. Results from the current study also are in agreement with Jofré et al. (2009) who observed 3.5 log unit reductions in Salmonella spp. populations inoculated on marinated beef loin following HPP at 600 MPa for 6 min, with minimal reductions of S. aureus (1 log unit) in cooked ham. The current results did not demonstrate sufficient E. coli O157:H7 and L. monocytogenes reductions to meet regulatory requirements; however, the HPP process used in this study may be suitable for processors seeking shelf life extension or use as an additional intervention to improve the safety of their product. In this study, two alternative jerky processing interventions were evaluated for reducing pathogen populations for use in a nontraditional jerky process. Data demonstrated that HPP could reduce several bacterial pathogens present on raw beef strips and jerky, but HPP would not be useful as single processing intervention. Alternatively, a boiling water (100 2 C) treatment proved to be effective at reducing pathogen populations and could be easily implemented by commercial jerky processors or home food preservers. It is also important to note that cell injury was not assessed in this study. Populations of pathogens exposed to HPP and boiling water treatments could be placed into an injured state rather than killed, and subsequently recover to a viable state if post-lethality processing conditions are suitable for recovery and growth. Further evaluation of cell injury and future validation of these processes in-plant could provide processors and regulators with alternative strategies for safe and shelf-stable jerky products. Additional sensory evaluation and determination of consumer acceptability of products subjected to HPP and boiling water treatment also are warranted for future studies. It is recommended that those commercial and home processors of jerky products follow the processing guidelines set forth by the USDA-FSIS, and if alternative processes are to be used, such as those evaluated in this study, those processes be scientifically validated to demonstrate necessary reductions in applicable pathogens as required by the USDA-FSIS.
Acknowledgments The authors would like to thank Christopher Gardner, Rebekah Miller, and Richard Swartz for their technical assistance with this research project. This project was funded, in part, by Lazy Jims Jerky Spice, the Department of Food Science, and College of Agricultural Sciences at the Pennsylvania State University. References Balasubramaniam, V. M., Farkas, D., & Turek, E. J. (2008). Preserving foods through high- pressure processing. Journal of Food Technology, Nov Ed, 32e38. Ferrini, G., Composada, J., Aranu, J., & Gou, P. (2012). Colour modification in a cured meat model dried by quick-dry-slice processÒ and high pressure processed as a function of NaCl, KCL, K-lactate and water contents. Innovative Food Science & Emerging Technologies, 13, 69e74. Garriga, M., & Aymerich, M. T. (2009). Advanced decontamination technologies: high hydrostatic pressure on meat products. In F. Toldrà (Ed.), Safety of meat and processed meat (pp. 183e188). New York, NY: Springer Science. Garriga, M., Aymerich, M. T., Costa, S., Monfort, J. M., & Hugas, M. (2002). Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage. Journal of Food Microbiology, 19, 509e518. Harrison, J. A., Harrison, M. A., Rose-Morrow, R. A., & Shewfelt, R. L. (2001). Homestyle beef jerky: effect of four preparation methods on consumer acceptability and pathogen inactivation. Journal of Food Protection, 64, 1194e1198. Jofré, A., Aymerich, T., Grébol, N., & Garriga, M. (2009). Efficiency of high hydrostatic pressure at 600 MPa against food-borne organisms by challenge tests on convenience meat products. LWT-Food Science and Technology, 42, 924e928. Keene, W., Sazie, E., Kok, J., Rice, D., Hancock, D., Balan, V., et al. (1997). An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat. Journal of the American Medical Association, 277, 1229e1231. Macfarlane, J. J. (1973). Pre-rigor pressurization of muscle: effects on pH, shear value and taste panel assessment. Journal of Food Science, 38, 294e298. Minnesota Department of Health (MDH). (2012). State issues consumer advisory for Hoffman Town and country meat market turkey jerky. Available at http://www. health.state.mn.us/news/pressrel/2012/salmonella082312.html Date last accessed 11/5/12. Moussa, M., Perrier-Cornet, J. M., & Gervais, P. (2006). Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli. Applied and Environmental Microbiology, 72, 150e156. Patterson, M. F. (2005). Microbiology of pressure-treated foods. Journal of Applied Microbiology, 98, 1400e1409. Porto-Fett, A., Call, J. E., Shoyer, B. E., Hill, D. E., Pshebniski, C., Cocoma, G. J., et al. (2010). Evaluation of fermentation, drying, and/or high pressure processing on viability of Listeria monocytogens, Escherichia coli O157:H7, Salmonella spp., and Trichinella spiralis in raw pork and genoa salami. International Journal of Food Microbiology, 140, 61e75. Rabatsky-Her, T., Dingman, D., Marcus, R., Howard, R., Kinney, A., & Mshar, P. (2002). Deer meat as the source for a sporadic case of Escherichia coli O157:H7 infection, Connecticut. Emerging Infectious Diseases, 8, 525e527. Richard, A., & Cutter, C. (2011). Validation of a pickling process for controlling pathogens associated with hard-cooked eggs. Journal of Food Safety, 31, 417e423. Roberts, W. A. (2003). Something to savor. Prepared Foods, 172, 25e30. Schaefer, R. (2013). SBDC national information clearinghouse: Beef jerky manufacturing. Available at http://www.sbdcnet.org/small-business-researchreports/beef-jerky-manufacturing accessed 01.05.13. Simonin, H., Duranton, F., & De Lambaallerie, M. (2012). New insights into the highpressure processing of meat and meat products. Comprehensive Reviews in Food Science and Food Safety, 11, 285e306. Sloan, A. (2011). Top 10 food trends. Journal of Food Technology24e40. Apr Ed. U.S. Centers for Disease Control (CDC). (1995). Outbreak of salmonellosis associated with beef jerky e New Mexico, 1995. Morbidity and Mortality Weekly Report, 44, 785e788. U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). (2012a). FSIS compliance guideline for meat and poultry jerky produced by small and very small establishments. Available at http://www.fsis.usda.gov/PDF/ Compliance_ Guideline_Jerky_2012.pdf Date last accessed 12.10.12. U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). (2012). Microbiological laboratory guidebook. Available at http://www.fsis.usda. gov/science/microbiological_lab_guidebook/Date last accessed 12.10.12.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
High-pressure processing and boiling water treatments for reducing Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus during beef jerky processing Joshua A. Scheinberg, Amanda L. Svoboda, Catherine N. Cutter* Department of Food Science, The Pennsylvania State University, University Park, 202 Food Science Building, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 31 October 2013 Accepted 1 November 2013
Beef jerky is a convenient, ready-to-eat meat product, but requires processing lethality steps to ensure the safety of the product. Previous outbreaks involving various jerky products have highlighted the risks associated with jerky and the importance of utilizing pathogen interventions during processing. In this study, two alternative interventions were evaluated for reducing pathogen populations during jerky processing. Results demonstrated that high pressure processing (HPP; two treatments of 550 MPa, 60 s) could produce significant (p < 0.05), but variable reductions (6.83 and 4.45 log10 CFU/strip) of Salmonella spp. and Escherichia coli O157:H7, respectively, on resulting beef jerky. HPP treatments, however, produced minor reductions (p < 0.05) of Gram-positive pathogens, resulting in reductions of 1.28 and 1.32 log10 CFU/strip of Listeria monocytogenes and Staphylococcus aureus, respectively. Alternatively, boiling water (100 2 C) treatments (20e30 s) used after marination and prior to dehydration, reduced Salmonella spp., E. coli O157:H7, L. monocytogenes, and S. aureus populations >5.0 log10 CFU/strip in resulting beef jerky. Thus, 20 or 30 s boiling water (100 2 C) treatments could be effective interventions for commercial jerky processors or home food preservers. Future validation of these processes in-plant could provide processors and regulators with alternative strategies for safe and shelfstable jerky products. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Jerky Pathogens High pressure processing Boiling water Intervention
1. Introduction Beef jerky and other dried meat snacks have continued to grow in popularity. In fact, these products are among the Top 10 Consumer Packaged Goods Categories, showing considerable growth in sales in the U.S. during the last decade (Roberts, 2003; Sloan, 2011). Although a few large beef jerky processors currently dominate 70% of U.S. jerky sales, small niche meat processors continue to make a wide range of unique jerky and dried meat snacks throughout the country (Schaefer, 2013). Commercially-processed, whole-muscle beef jerky is a convenient, ready-to-eat (RTE) meat product, but requires sufficient processing lethality steps to ensure the safety of the product throughout its shelf life. Previous outbreaks involving various jerky products have highlighted the risks associated with meat jerky and the importance of validating jerky processing methods. The most common pathogen associated with beef jerky outbreaks has been Salmonella, with the largest outbreak occurring in 1994, affecting 93 persons who consumed beef jerky made in
* Corresponding author. Tel.: þ1 814 865 8862; fax: þ1 814 863 6132. E-mail address: [email protected] (C.N. Cutter). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.11.002
New Mexico (CDC, 1995). A more recent outbreak in 2012 involved 4 cases of salmonellosis from individuals who consumed turkey jerky made in Minnesota (MDH, 2012). Additionally, consumption of venison jerky has resulted in infections with Escherichia coli O157:H7 in Oregon in 1994 and Connecticut in 2002 (Keene et al., 1997; Rabatsky-Her et al., 2002). In an effort to control spoilage and pathogenic microorganisms during jerky processing, many processors have sought alternative intervention methods. High pressure processing (HPP) is one such method that has seen a rise in its use among meat processors. HPP was first used on meat products by Macfarlane (1973) to increase the tenderness of meat; however its lethal effect on bacteria is considered an important reason to use this technology (Garriga & Aymerich, 2009; Simonin, Duranton, & De Lambaallerie, 2012). Cell death by HPP is achieved through a combination of effects, with cell membrane alteration as a primary cause of cellular death (Moussa, Perrier-Cornet, & Gervais, 2006; Patterson, 2005; Simonin et al., 2012). Using HPP, variable pathogen reductions have been demonstrated on several dried meat products. Jofré, Aymerich, Grébol, and Garriga (2009) demonstrated a >3.0 log unit reduction of Listeria monocytogenes and Salmonella spp. on dried cured ham when HPP was applied at 600 MPa for 6 min Porto-Fett et al.
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(2010) observed a >5 log unit reduction of E. coli O157:H7 on dried salami when treated with 600 MPa for up to 5 min. To our knowledge, no published study has examined the effects of HPP on beef jerky products experimentally inoculated with pathogens. Although generally regarded as an expensive alternative to other antimicrobial interventions, the use of HPP in the food industry has become more widespread in the last decade (Patterson, 2005). Custom HPP systems range between $500,000 and $2.5 million; however numerous facilities across the U.S. now offer HPP services (Balasubramaniam, Farkas, & Turek, 2008; Patterson, 2005). Although HPP service costs will vary due to different operating parameters, product specifications, packaging, volume, and labor costs, available sources have estimated treatment costs ranging between $0.04 and $0.11 per pound of product (Balasubramaniam et al., 2008; Patterson, 2005). Depending on the resources and product specifications, HPP may be a useful and feasible antimicrobial intervention for the jerky processor. Nevertheless, many jerky processors have sought other alternative processes and focused on improvements to marinade recipes or altered heat treatments, rather than utilize processes like HPP. One such strategy is to heat raw meat strips in a boiling marinade prior to dehydration. Harrison, Harrison, Rose-Morrow, and Shewfelt (2001) demonstrated >5 log unit reduction of E. coli O157:H7, L. monocytogenes, and Salmonella after beef strips were marinated and boiled for 5 min in the marinade, prior to dehydration. However, the 5 min boiling method produced significant changes to the color and saltiness of the finished jerky compared to jerky that had only been marinated and dehydrated. It is unknown whether boiling treatments of less than 5 min prior to dehydration would be as effective in reducing pathogens. It is also unknown whether variations in marinade recipes would alter the effectiveness of this boiling marinade technique, and whether they may contribute to the sensory changes. The use of boiling water and minimum boiling water exposure times (<1 min), rather than a boiling marinade for several minutes, may offer a more suitable and consistent alternative for jerky processors seeking additional lethality processes that produce minimal sensory changes to the final jerky product. Jerky processed and sold commercially is regulated by the USDA-Food Safety and Inspection Service (USDA-FSIS). Currently, the USDA-FSIS Jerky Compliance Guidelines provide recommended time, temperature, and relative humidity (RH) processing parameters to achieve necessary reductions in selected pathogens for jerky processing (USDA-FSIS, 2012a). The USDAFSIS recommends that current jerky processing methods should achieve 5 log unit reductions of Salmonella spp., E. coli O157:H7, and at least a 3 log unit reduction of L. monocytogenes in the final jerky product (USDA-FSIS, 2012a). Many small and very small processors may choose to produce jerky products that may not follow typical processing parameters as recommended by the USDA-FSIS. In these instances, scientific support must be provided to demonstrate that the processes used produce the necessary reductions of Salmonella spp., E. coli O157:H7, and L. monocytogenes in the final jerky product. Due to the increasing popularity of home jerky processing and the production of unique jerky products by processors who may not utilize typical dehydration times and temperatures, as recommended by the USDA-FSIS, the purpose of this study is to investigate the efficacy of two alternative antimicrobial interventions (HPP and submersion in boiling water (100 2 C)), for use in applications where standard heat and drying lethality treatments for jerky processing may not be implemented. The parameters used in this study mimic those selected by a very small jerky processor seeking additional antimicrobial interventions for their nontraditional jerky product.
2. Materials and methods 2.1. Preparation of bacterial inoculum Cultures of Salmonella Typhimurium ATCC 14028, Salmonella Enteritidis ATCC 13076, E. coli O157:H7 ATCC 43889, L. monocytogenes Scott A, and a human Staphylococcus aureus isolate obtained from the Food Microbiology Culture Collection at The Pennsylvania State University Food Science Department were used to produce the inoculum cocktail. All cultures were stored at 80 C in tryptic soy broth (TSB; Becton Dickinson and Company; BD, Sparks, MD) supplemented with 20% (v/v) glycerol to ensure viability. Pathogen cultures were prepared by transferring the frozen stock to fresh TSB and incubating at 37 C for 24 h. Colonies were later isolated by streaking TSB cultures to tryptic soy agar (TSA; BD). Single colonies of each pathogen were isolated and transferred to fresh TSB and repeated sequentially in triplicate to ensure pure culture. Isolated colonies of each pathogen were confirmed using Gram stains and Salmonella spp., L. monocytogenes, Staphylococcus aureus, and E. coli O157:H7 agglutination kits (Remel, Lenexa, KS; Oxoid, Basingstoke, Hampshire) prior to inoculum preparation. Inoculum was prepared using the methodologies described by Richard and Cutter (2011), in which single colonies of each pathogen were transferred to 500 mL of TSB, in triplicate, and grown statically at 37 C for 24 h to obtain cell concentrations of 9 log10 CFU/ml. As was also described in Richard and Cutter (2011), equal volumes of each pathogen culture (1500 ml each) were mixed in a large sterilized metal bin to produce a non-diluted cocktail inoculation bath for the raw beef strip inoculation. This inoculum preparation allowed for the simultaneous inoculation of large quantities of beef samples. 2.2. Preparation and inoculation of raw beef strips Beef top rounds were purchased from a local supermarket and transported in an insulated cooler with ice to the Penn State Muscle Foods Microbiology Laboratory and stored at 20 C until further use. Frozen beef was thawed for approximately 24 h at 4 C to soften the meat, aseptically removed from the packaging, and all visible fat was removed with a sterile knife. Beef strips were prepared using a sanitized mechanical meat slicer (Globe; Dayton, OH) producing 3.2 mm thick, uniform pieces. Each beef strip was trimmed to 10.2 cm 5.0 cm, packaged in sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI), and stored at 4 C until fully defrosted. For both the HPP and boiling water submersion experiments, prepared beef strips (n ¼ 70 for HPP treatment; n ¼ 57 for boiling water treatment) were placed aseptically into the inoculum bath for 30 min under a biological safety hood with frequent stirring and agitation. Following inoculation, each beef strip was removed from the inoculum aseptically using sterile forceps, allowed to drip for 3e5 s, and placed onto sterile drip trays. Beef strips were allowed to dry on the drip trays under the biological safety hood for 15 min prior to each jerky experiment. After air-drying, beef strips were placed into a bowl of pre-prepared marinade solution, mixed well, covered with plastic wrap, and allowed to marinate for 24 h at 4 C. Following marination, beef strips were removed from the marinade, allowed to drip for 3e5 s, and placed onto sterile drip trays under the biological safety hood for 15 min prior to further treatments. The marinade solution was prepared by mixing 57 g of a proprietary spice blend (Lazy Jims Jerky Spice; York Springs, PA) with 237 ml of sterile water for every 454 g (1 pound) of beef. This solution was heated to 83 C and cooled to 25 C prior to use, in accordance with the recipe and methods provided by the manufacturer.
J.A. Scheinberg et al. / Food Control 39 (2014) 105e110
2.3. Boiling water submersion treatments Inoculated and marinated raw beef strips used for boiling water submersion treatments (n ¼ 57) were aseptically transferred to a sterilized, metal wire basket (n ¼ 9 per basket/time point), covered with foil, and stored aseptically at 4 C prior to the boiling water treatment. Four liters of sterile water was prepared for each of the boiling water exposure time trials (0, 5, 10, 15, 20, 30 s). In each trial, 4 L of sterile water was poured into a sterile metal bin sitting on two pre-heated laboratory hot plates (25.4 cm 25.4 cm; VWR, NY), covered with foil, and heated until boiling (100 2 C). Each metal basket (one per time point) containing the inoculated and marinated beef strips was lowered into the boiling water (100 2 C), ensuring all strips were submerged, and held for the respective time. Since the temperature of the water typically dropped below boiling after the addition of the beef strips, the exposure time was measured once boiling resumed, 15 s. Following the boiling treatment, baskets were removed from the water and beef strips aseptically transferred onto dehydrator drying racks, and allowed to dry in the biological safety hood for 15 min. Boiled beef strips were then dehydrated using a 9-tray dehydrator (Excalibur Dehydrators, Fort Lauderdale, FL) and dried at a temperature of 55 C for 4 h which was contained in a biological safety hood with drying racks rotated each hour. The temperature of the dehydrator was verified to be at 55 C prior to the dehydration process to account for dehydrator come up time. Temperatures were monitored every 30 min using a thermometer (VWR) placed inside the dehydrator, and taken at several locations within the dehydrator to ensure beef slices were exposed to the same temperature. Relative humidity (RH) measurements were also taken every hour, although the Excalibur Dehydrator is not designed to control RH. Temperatures fluctuated in certain areas by 1e5 C. Internal jerky temperatures were verified to have reached 55 C following the 4-h dehydration cycle. After dehydration, beef jerky slices were placed into vacuum packaging bags (15.2 cm 20.3 cm; Prime Source, Kansas City, MO) and vacuum sealed (3 pieces of jerky/bag) using an Ultravac UV-250 vacuum sealer (UltraSource USA, Kansas City, MO). Raw beef strips and jerky subjected to boiling water (100 2 C) submersion treatments were sampled for microbiological analysis following each of the steps (n ¼ 3 beef strips or jerky per processing step and time point): inoculation, marination, dehydration, 1 h following vacuum packaging, and after 7 and 14 days of vacuum packagedstorage at room temperature (22e25 C).
transferred to a filtered stomacher bag (Interscience, St. Rockland, MA), weighed, and diluted with 10 times the volume of the sample with Buffered Peptone Water (BPW; BD) and stomached for 2 min at 230 rpm (Stomacher 400; Seward, West Sussex, UK). The stomachate was serially diluted in BPW and aliquots of 0.1 ml and 0.5 ml were spread plated in duplicate and quadruplicate, respectively, onto selective agars to achieve a detection limit of 1 log10 CFU/strip. Selective agars used for the enumeration of surviving pathogens included: Xylose Lysine Deoxycholate agar (XLD; BD), Baird-Parker agar with egg yolk tellurite (Remel), Cefixime Tellurite-Sorbitol MacConkey agar (CT-SMAC; Remel), and Modified Oxford agar with antibiotic supplement (MOX; Remel) for enumeration of Salmonella spp., S. aureus, E. coli O157:H7, and L. monocytogenes populations, respectively. Samples also were transferred to enrichment media to determine the presence or absence of surviving cells in those samples with low pathogen concentrations. Pathogens were enriched using methods adopted from the USDA-FSIS Microbiological Laboratory Guidebook (MLG; USDA-FSIS, 2012a, 2012b). Briefly, enrichment of Salmonella spp. utilized a non-selective preenrichment of lactose broth (BD), followed by selective enrichments of tetrathionate broth (BD) and selenite cystine broth (BD) with subsequent plating on XLD. UVM (BD) and Fraser broths (BD) were used as primary and secondary enrichments, respectively, for L. monocytogenes. Following the secondary enrichment step, samples were plated on MOX (BD). Gram-negative broth (GN; BD) and TSB w/5% NaCl (TSB; BD) were used as primary enrichments for E. coli O157:H7 and S. aureus, with subsequent plating on CT-SMAC and Baird-Parker agar, respectively. Measurements of pH and water activity (Aw) of all jerky samples were determined at each sampling time using a VWR Symphony pH meter (VWR) and Aqua Lab 4TE (Decagon Devices, Pullman, WA) water activity meter. 2.6. Statistical analysis All bacterial populations were converted to log10 CFU/strip for analysis and transformation of data was performed as necessary to meet the assumptions of the statistical test. Analyses of statistical tests were performed using SAS (SAS software, Version 9.3, SAS Institute Inc., Cary, NC). In cases where there was a zero count on duplicate plates, a count of 1 CFU was assigned to allow for count conversion to log10 CFU/strip. Comparisons of pathogen populations were determined using ANOVA with Tukey’s HSD test at a ¼ 0.05.
2.4. HPP treatment
3. Results
Inoculated and marinated raw beef strips used for HPP treatments (n ¼ 70) were immediately transferred to dehydrator drying racks, dehydrated using a 9-tray dehydrator, and vacuum packaged, as described in section 2.3. HPP was performed using the vertically loaded QFP 2L-700 High Pressure Laboratory Food Processing System (Avure, Franklin, TN) capable of operating at 100,000 PSI (689 MPa). Each vacuum packaged bag of beef jerky samples were high pressure-treated at 550 MPa for two consecutive, 60 s treatments at a process water temperature of 22 C. Raw beef strips and jerky used for the HPP treatments were sampled for microbiological analysis following each of the steps (n ¼ 10 beef strips or jerky per each processing step): inoculation, marination, dehydration, 1 h following vacuum packaging, HPP, and 7 and 14 days following vacuum-packaged storage at room temperature.
3.1. Boiling water submersion treatments
2.5. Microbiological analyses For both the HPP and boiling water submersion experiments, each individual raw beef strip and jerky were aseptically
107
Boiling water (100 2 C) treatments produced variable reductions in pathogen populations on beef slices (Figs. 1e6). Interestingly, populations of Salmonella spp., exposed to any of the boiling water treatments and subsequent dehydration, were reduced >5 log10 CFU/strip from pre-marination levels. Conversely, E. coli O157:H7, L. monocytogenes, and S. aureus populations were not reduced >5 log10 CFU/strip until after exposure to 20 and 30 s of boiling water and dehydration. As a control, beef slices not exposed to the boiling water treatment (0 s) were briefly immersed in water (20e22 C) to observe the isolated effects of the other processes (marinating, dehydration, and vacuum packaging). Control samples demonstrated reductions (<3 log10 CFU/strip) in E. coli O157:H7, L. monocytogenes, and S. aureus populations. Salmonella spp. populations were reduced 5 log10 CFU/strip after marination, dehydration, vacuum-packaging, and 24 h of vacuum-packaged storage. Boiling water treatments of 20 and 30 s resulted in log reductions >5 log10 CFU/strip after vacuum-packaging for all pathogens tested
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9
9
8
8
7
7
6 5 4
EC
6
EC
SS
5
SS
SA
3
LM
2
4
SA
3
LM
2 1
1
0
0
Inoculation
Boil
Dehydration
Inoculation
Vacuum
Fig. 1. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (0 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
Boil
Dehydration
Vacuum
Fig. 4. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (15 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
9 8 7 6
EC
5
SS
4
SA
3
LM
2 1 0
Inoculation
Boil
Dehydration
Vacuum
in this study, with Salmonella spp. reduced to levels below the detection limit and also negative for the pathogen following enrichment. Minimal changes in pH were observed among all treatments throughout the experiment; with a slight increase following marination and a final pH of 6.54 among 30 s treatment samples (Table 1). RH measurements taken during dehydration ranged from 10.4 to 52.7% on the low and high ends. Interestingly, initial aw levels of 0.98 among inoculated samples were reduced to 0.57 after 0 s treatments and to 0.64 among 30 s treatments, exhibiting less aw reduction as boiling water treatment time increased. These differences observed between final aw levels between each
9 8 7 6
EC
5
SS
4
SA LM
2
EC SS SA LM
* Inoculation
Fig. 2. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (5 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
3
9 8 7 6 5 4 3 2 1 0
1 0
Boil
Dehydration
Dehydration
Vacuum
Fig. 5. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (20 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation. * Pathogen populations were reduced below the detection limit and were negative for enrichment.
treatment, could suggest that longer boiling water exposures may produce a greater dilution of solutes present on marinated beef strips. 3.2. HPP treatments As expected, marination, dehydration, and vacuum packaging alone, did not result in >5 log10 CFU/strip reductions of the pathogens evaluated in this study (Table 2). Although reduction in aw occurred following dehydration (from aw of 0.97 to 0.74), these
9 8 7 6 5 4 3 2 1 0
EC SS SA LM
* Inoculation
Inoculation
Boil
*
Boil
Dehydration
* Vacuum
Vacuum
Fig. 3. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (10 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation.
Fig. 6. Average counts (log10 CFU/strip) of E. coli O157:H7 (EC), Salmonella spp. (SS), S. aureus (SA), and L. monocytogenes (LM) during jerky processing using a (30 s) boiling water treatment. Error bars indicate the mean (n ¼ 3) value one standard deviation. * Pathogen populations were reduced below the detection limit and were negative for enrichment.
J.A. Scheinberg et al. / Food Control 39 (2014) 105e110
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Table 1 pH and aw values of beef samples during jerky processing and boiling water treatments.a Boiling water exposure times
Inoculation
Marinationb
Boiling water Treatment
0s
5.60 0.07 0.982 0.02 e
6.60 0.06 0.971 0.03 e
10 sc
e
e
c
e
e
20 sc
e
e
30 sc
e
e
6.90 0.957 6.93 0.954 6.86 0.951 6.89 0.940 6.70 0.943 6.63 0.954
5 sc
15 s
Dehydration
0.02 0.003 0.13 0.006 0.01 0.007 0.01 0.007 0.07 0.003 0.02 0.002
6.54 0.642 6.64 0.619 6.67 0.601 6.70 0.568 6.67 0.642 6.71 0.628
0.07 0.02 0.06 0.03 0.01 0.01 0.06 0.03 0.08 0.01 0.03 0.01
Vacuum package 6.54 0.577 6.67 0.581 6.57 0.616 6.52 0.611 6.68 0.629 6.54 0.649
0.07 0.04 0.04 0.02 0.07 0.01 0.07 0.02 0.06 0.006 0.08 0.008
Note: Analysis of pH and aw was performed immediately following each of the experimental steps (Inoculation, Marination, Boiling Water Treatment, Dehydration, 1 Hour Following Vacuum Packaging). a pH and aw values are mean of (n ¼ 3) for each processing step, pH values are listed on top of aw values for each processing step and exposure time. b pH of marinade solution was 6.70. c All beef strips (n ¼ 57) were inoculated and marinated together in the same containers, pH and aw was performed (n ¼ 3) following inoculation and marination which represented conditions of all beef strips prior to being separated into groups based on boiling water exposure times. Values listed for inoculation and marination at (0 s) are the same for the other exposure times, represented by a ().
jerky sensory characteristics (Harrison et al., 2001). In this study, boiling water (100 2 C) treatments of 30 s were selected to evaluate the efficacy of a rapid and easy method for pathogen reduction that could be employed by both commercial jerky processors or by the home food preserver. Our results demonstrate that the application of a 20e30 s boiling water submersion followed by dehydration and vacuum packaging can reduce pathogen populations (Salmonella spp., E. coli O157:H7, L. monocytogenes, and S. aureus) > 5 log10 CFU/strip. As such, this step may be suitable for meeting the lethality target recommended by current USDA-FSIS jerky processing guidance. It should additionally be noted that jerky exposed to boiling water treatments exhibited a final aw of 0.57e0.65 and a pH of 6.52e6.68 depending on the boiling water treatment time, which may be lower or higher than generally desired by certain processors. Current USDA-FSIS guidance recommends jerky be processed to achieve a final aw of 0.85 if packaged in an aerobic environment, although a critical limit of 0.91 could be acceptable if vacuum packaged (USDA-FSIS, 2012a). Current USDA-FSIS guidance does not provide specific pH values to be achieved in marinades or final jerky products, although it is recognized that pH is a critical parameter that can provide additional product safety but will vary depending on the process. The efficacy of HPP to reduce pathogens in food products has been studied extensively. However, little to no data exists on the effects of HPP for reducing bacterial pathogens associated with beef jerky. Due to the increase in HPP facilities around the country, processors of dried foods like beef jerky, could utilize HPP as an alternative lethality process to minimize the heat and dehydration treatments performed during commercial jerky processing. To date,
changes did not appear to drastically reduce pathogen populations; however reductions were statistically significant (p < 0.05). RH measurements taken during dehydration ranged from 8.2 to 51.5% on the low and high ends. HPP treatments of two cycles of 550 MPa for 60 s, were performed immediately following vacuum packaging of the dehydrated jerky, resulting in variable reductions in pathogen populations. The 2-cycle HPP treatment was found to have the greatest effect on Salmonella spp. populations with reductions of 3.2 log10 CFU/strip. It is important to note that reductions on individual samples were variable. Small reductions of L. monocytogenes (1.2 log10 CFU/strip) and S. aureus (1.3 log10 CFU/ strip) populations also were observed following HPP treatments; however, E. coli O157:H7 populations did not significantly change as an immediate result of HPP. Fourteen day storage (20e22 C) of HPP-processed samples resulted in further reductions in Salmonella spp. (1.4 log10 CFU/strip), and E. coli O157:H7 (1 log10 CFU/strip) populations. Populations of L. monocytogenes and S. aureus increased slightly following 14 day vacuum packaged storage; however these increases were likely due to higher variation among jerky samples. Interestingly, total log reductions of >5 log CFU/strip were observed in Salmonella spp. over the course of the experiment, with E. coli O157:H7 reductions reaching 4.4 log10 CFU/strip after 14 days of vacuum packaged storage. 4. Discussion Previous research utilizing applications of a heat treatment, prior to dehydration, has demonstrated variable effects on the reduction of pathogens, while also producing significant changes in
Table 2 Average counts (log10 CFU/strip) of L. monocytogenes, Salmonella spp., E. coli O157:H7, and S. aureus; pH and aw during jerky processing and subjected to HPP. Inoculation L. monocytogenes Salmonella spp. E. coli O157:H7 S. aureus pHx a wx
7.85 7.43 7.30 7.25 5.60 0.982
0.14a 0.10a 0.18a 0.14a 0.08 0.003
Marinationz 7.61 7.15 6.48 7.15 6.68 0.971
0.25a 0.19a 0.22b 0.40ab 0.07 0.003
Dehydration 6.85 5.57 6.12 6.66 6.71 0.735
0.17bc 0.40b 0.98b 0.37bc 0.02 0.05
Vacuum package 6.71 5.33 4.21 6.16 6.90 0.784
0.19c 0.33b 0.95c 0.39ce 0.03 0.07
HPP 5.51 2.07 3.82 4.83 6.77 0.751
HPP (7d)
0.19d 1.28c 0.89c 0.37d 0.04 0.02
6.49 1.60 3.41 6.01 6.82 0.78
0.63ce 0.96c 0.6d 0.5e 0.09 0.05
HPP (14d)
Log10 CFU/strip Reductiony
6.57 0.47bce 0.6 0.57d 2.85 0.49e 5.93 0.29c 6.87 0.03 0.61 0.02
1.28 6.83 4.45 1.32
Note: Log10 CFU/strip counts and standard deviations are mean of (n ¼ 10; individual beef strips or jerky) for each processing step. Microbiological and pH and aw analysis was performed immediately following each of the experimental steps (Inoculation, Marination, Dehydration, 1 h Following Vacuum Packaging, HPP, HPP (7 d), HPP (14 d)). aee Different letters represent significant difference between processing steps within each pathogen (p < 0.05). x pH and aw values are mean of (n ¼ 3) for each processing step. y Log10 CFU/strip reduction was calculated by subtracting populations subjected to HPP (14 d) from initial, untreated inoculation counts. z pH of marinade solution was 6.70.
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research on HPP treatments of dried meat products has demonstrated bacterial pathogen reductions when utilizing pressures >550 MPa for >5 min. At these pressures and times, significant changes in meat color and water content also have been documented (Ferrini, Composada, Aranu, & Gou, 2012). In the current study, minimal HPP exposures (2 treatments of 60 s) were used in an effort to minimize changes in sensory characteristics (color, texture, etc.) of the beef jerky, although those effects were not investigated. The results of this study demonstrated significant reductions (p < 0.05) in all pathogens exposed to the HPP treatments. However, only Salmonella spp. populations were reduced enough to satisfy USDA-FSIS regulatory requirements. E. coli O157:H7 levels were not reduced by 5 log10 CFU/strip, although HPP treatment did significantly affect the pathogen, resulting in 4.4 log10 CFU/strip reductions over the 14 day experiment. Alternatively, HPP was less effective against the Gram-positive pathogens, since S. aureus and L. monocytogenes were minimally reduced by 1.0 log10 CFU/strip at day 14. Garriga, Aymerich, Costa, Monfort, and Hugas (2002) observed similar results, reporting recovery (3 log units) of L. monocytogenes and S. aureus during up to 20 day storage of inoculated, cooked ham slurry exposed to 400 MPa for 10 min. Results from the current study also are in agreement with Jofré et al. (2009) who observed 3.5 log unit reductions in Salmonella spp. populations inoculated on marinated beef loin following HPP at 600 MPa for 6 min, with minimal reductions of S. aureus (1 log unit) in cooked ham. The current results did not demonstrate sufficient E. coli O157:H7 and L. monocytogenes reductions to meet regulatory requirements; however, the HPP process used in this study may be suitable for processors seeking shelf life extension or use as an additional intervention to improve the safety of their product. In this study, two alternative jerky processing interventions were evaluated for reducing pathogen populations for use in a nontraditional jerky process. Data demonstrated that HPP could reduce several bacterial pathogens present on raw beef strips and jerky, but HPP would not be useful as single processing intervention. Alternatively, a boiling water (100 2 C) treatment proved to be effective at reducing pathogen populations and could be easily implemented by commercial jerky processors or home food preservers. It is also important to note that cell injury was not assessed in this study. Populations of pathogens exposed to HPP and boiling water treatments could be placed into an injured state rather than killed, and subsequently recover to a viable state if post-lethality processing conditions are suitable for recovery and growth. Further evaluation of cell injury and future validation of these processes in-plant could provide processors and regulators with alternative strategies for safe and shelf-stable jerky products. Additional sensory evaluation and determination of consumer acceptability of products subjected to HPP and boiling water treatment also are warranted for future studies. It is recommended that those commercial and home processors of jerky products follow the processing guidelines set forth by the USDA-FSIS, and if alternative processes are to be used, such as those evaluated in this study, those processes be scientifically validated to demonstrate necessary reductions in applicable pathogens as required by the USDA-FSIS.
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