Effects of High-Pressure Processing on the Safety, Quality, and Shelf Life of Ready-to-Eat Meats

Transcription

1 1709 Journal of Food Protection, Vol. 67, No. 8, 2004, Pages Copyright, International Association for Food Protection Effects of High-Pressure Processing on the Safety, Quality, and Shelf Life of Ready-to-Eat Meats MELINDA M. HAYMAN, 1 IRENE BAXTER, 2 PATRICK J. O RIORDAN, 2 AND CYNTHIA M. STEWART 1 * 1 Food Safety and Quality and 2 Supply Chain Innovation Groups, Food Science Australia, P.O. Box 52, North Ryde NSW 1670, Australia MS : Received 31 December 2003/Accepted 22 March 2004 ABSTRACT Ready-to-eat (RTE) meats (low-fat pastrami, Strassburg beef, export sausage, and Cajun beef) were pressure treated at 600 MPa, 20 C, for 180 s to evaluate the feasibility of using high-pressure processing () for the safe shelf-life extension of these products. After processing, samples were stored at 4 C for 98 days during which time microbiological enumeration and enrichments were performed. Additionally, sensory analyses were undertaken to determine consumer acceptability and purchase intent over the duration of storage. Counts of aerobic and anaerobic mesophiles, lactic acid bacteria, Listeria spp., staphylococci, Brochothrix thermosphacta, coliforms, and yeasts and molds revealed that there were undetectable or low levels for all types of microorganisms throughout storage. Comparison of consumer hedonic ratings for unprocessed and processed meats revealed no difference in consumer acceptability, and no deterioration in the sensory quality was evident for any of the products tested during the study. Additionally, inoculated pack studies were conducted to determine if could be used as a postlethality treatment to reduce or eliminate Listeria monocytogenes and thus assess the potential use of in a hazard analysis critical control point plan for production of RTE meats. Inoculated samples (initial level of 10 4 CFU/g) were pressure treated (600 MPa, 20 C, for 180 s) and stored at 4 C, and survival of L. monocytogenes was monitored for 91 days. L. monocytogenes was not detected by plating methods until day 91, but selective enrichments showed sporadic recovery in three of the four products examined. The results show that at 600 MPa, 20 C, for 180 s can extend the refrigerated shelf life of RTE meats and reduce L. monocytogenes numbers by more than 4 log CFU/g in inoculated product. High-pressure processing () is a nonthermal method of food preservation that has attracted much interest in the last two decades for its ability to inactivate microorganisms while maintaining the fresh-like qualities of many food products. Typically, pressures of 300 to 700 MPa are used to extend the shelf life and improve the safety of foods (11, 25). has several benefits over thermal processing of food, including the fact that pressure is transmitted instantaneously and uniformly, so it is evenly treated throughout (15, 23). Pressure, unlike heat, does not disrupt covalent bonds, so many of the nutrient and flavor compounds of the food are left intact, resulting in a product that often has a superior taste, nutritional value, and quality compared with thermally processed counterparts (4). The use of presents a number of potential benefits for preservation and modification of foods. Of particular interest are those that tend to prolong the shelf life of ready-to-eat (RTE) refrigerated foods by inactivating spoilage and pathogenic microorganisms. Several foods are currently available on the international market, including pressurized sliced ham in Spain; guacamole, salsa, juices, RTE meats, and oysters in the United States; jellies in Japan; and juice and fruit smoothie products in several European countries (9, 25). Additionally, seafood products are available in Australia. In response to the growing demand for convenience foods in recent years, there has been a growing market for * Author for correspondence. Tel: ; Fax: ; cindy.stewart@csiro.au. prepacked, sliced, RTE processed meat products. Meat and meat products are perishable and often have a limited shelf life (12). Manufacturing of these types of products involves slicing and packaging operations that take place after thermal treatment and therefore have a direct impact on their shelf life and safety (18). Processed meat products, such as deli meats, turkey frankfurters, and hot dogs, have been implicated in several outbreaks of listeriosis in the United States (8, 28, 32), and two significant outbreaks, which were traced back to pate and smoked mussels, have occurred in Australia (27). Listeria monocytogenes is a widely spread environmental microorganism that is able to survive and/or grow in many foods during refrigerated storage; modified atmospheres and vacuum packaging do not significantly affect growth (31). The microorganism frequently enters the human food supply and has been isolated from many foods, including milk, cheese, vegetables, fish, and other seafood, as well as from raw and processed meat and poultry (3, 10, 28). L. monocytogenes is a frequent contaminant of raw materials, can be continually reintroduced into the manufacturing environment, and is extremely difficult to exclude from food processing facilities (7, 22). For example, the incidence of L. monocytogenes in raw meat and poultry in a farm environment may be as high as 30 to 50% (14). Moist, refrigerated conditions, often found in processing environments, allow for survival and growth of this microorganism (30). Hygiene within the plant is important in limiting the contamination of processing equipment (22).

2 1710 HAYMAN ET AL. J. Food Prot., Vol. 67, No. 8 The risk of product contamination by L. monocytogenes can be reduced, but with current technology, the microorganism cannot be eradicated from the finished product environment (28). The International Commission on Microbiological Specifications for Foods has suggested that recontamination after cooking is the most common reason for the presence of L. monocytogenes in packaged, cooked sausages, such as frankfurters (13). If recontamination is assumed to be 10 CFU/g, then an in-package pasteurization treatment (such as ) could be applied as a means to achieve a 4-log reduction of L. monocytogenes and still meet a performance criterion of 10 3 CFU/g (1 CFU/kg) (13). L. monocytogenes has been estimated by the Centers for Disease Control and Prevention to cause 2,493 illnesses, 2,322 hospitalizations, and 499 deaths per year in the United States, 99% of which are via consumption of contaminated foods (20). Although the number of cases of listeriosis per year is relatively small, L. monocytogenes accounts for 28% of estimated food-related deaths in the United States, which is second only to Salmonella (31%). There are approximately 60 cases of listeriosis in Australia annually, with a mortality rate of 23% (6). The ability of L. monocytogenes to cause severe illness and death in young, elderly, and pregnant women and in immunocompromised people means it is important to eliminate or reduce numbers in the food supply, particularly in RTE foods. The contamination of RTE products has garnered great concern by processors and consumers, and the implementation of novel techniques, alone or in tandem with traditional methods, may help the food industry address this concern in a timely and cost-effective manner (19). One of the potential applications of is as an in-package cold pasteurization step for packaged RTE meats that may have been contaminated through portioning, slicing, comminuting, and/or packaging. combined with vacuum packaging and subsequent refrigerated storage has potential for a wide range of meat products, including cured meats, RTE meats or meals, and mechanically deboned meats. Although there are already commercial RTE meat products on the market in Europe and the United States, there is little information available in the scientific literature about and its ability to reduce pathogenic and spoilage microorganisms in packaged, RTE meats and the subsequent effect on extending the refrigerated shelf life of this type of product (19). The objective of the study was to investigate the feasibility of using to extend shelf life and improve the safety of refrigerated RTE meats. L. monocytogenes was selected as the target pathogen because of its frequent association with processed meats. The target performance criterion for L. monocytogenes in a RTE, packaged meat could be set at a 4-log reduction by pressure treatment based on published risk assessments (5, 13). Therefore, kinetic inactivation studies and product challenge testing were used to determine the required processing time at 600 MPa to achieve a 4-log reduction. The effect of in-package pressure treatment on the refrigerated shelf life of RTE meats with regard to the microbial safety and quality and the sensory character of the products was also investigated. MATERIALS AND METHODS Bacterial cultures. Eight L. monocytogenes strains (2542, 2655, 2657, 2340, 2341, 2342, 2343, 2345) isolated from processed meats or foods containing processed meats, as well as L. monocytogenes Scott A (2472), were selected from the Food Science Australia culture collection to be screened for their relative resistance to. Each strain was initially identified as L. monocytogenes using colony morphology on tryptic soy agar (TSA), Gram stain, catalase production, and a positive CAMP test result. Final confirmation was obtained using Listeria API strips (biomérieux, Marcy l Etoile, France). In preparation for all experiments, one loopful of cells was transferred from glycerol stocks (stored at 80 C) to tryptic soy broth (TSB, 10 ml; Oxoid, Basingstoke, England) and incubated for18hat37 C. Cells (100 l) were transferred to prechilled TSB (ph 6.3 adjusted with 1 M HCl, 50 ml), and incubated for 72hat15 C in a shaking water bath (45 strokes per min; U07030, Lauda, Königshofen, Germany), resulting in early-stationaryphase cells (approximately CFU/ml). Screening of L. monocytogenes strains for relative pressure resistance. Stationary-phase cells (500 l) were inoculated into fresh TSB (ph 6.3, 9.5 ml) to give approximately 10 8 CFU/ml. Samples (500 l) were transferred to 50 ml of fresh TSB (ph 6.3) to obtain approximately 10 6 CFU/ml. This was repeated for each strain. The culture (5 ml) was dispensed into sterile tubes (transfer plastic pipettes with half the stem cut off; Copan Italia, Brescia, Italy), and tubes were sealed by heating in a flame and crimping with pliers. The tubes were placed into plastic bags (205 by 350 mm, Cryovac, Melbourne, Australia) filled with 5,000 ppm of peroxyacetic acid (Peroxitane Sanitiser, Solvay Intertox, Sydney, Australia) and heat sealed immediately before pressure treatment. Samples were treated at 600 MPa for 0, 10, 20, 30, 40, and 60 s in a 2-liter pressure unit (Avure Technologies Inc., Seattle, Wash.). The time to reach 600 MPa (come-up time) was approximately 10 s, and pressure release after the indicated hold times was less than 2 s. All was conducted at ambient temperature (approximately 20 C). After pressure treatment, the cells were immediately enumerated by serial dilutions in 0.1% peptone water (Amyl Media, Dandenong, Australia). The samples were plated on tryptic soy yeast extract agar (TSYEA, ph 7.4; 30 g of TSB [Oxoid], 6 g of yeast extract [Amyl Media], 14 g of agar [Leiner Davis Gelatin, Sydney, Australia]) and the plates incubated at 37 C for 48 h. Two replicate experiments were conducted for each strain examined. Determination of effect of NaCl on pressure inactivation of L. monocytogenes Since L. monocytogenes strain 2542 (originally isolated from salami) was the most pressure resistant of the nine strains tested, it was selected for further screening experiments to determine the effect of NaCl on resistance to. NaCl levels were chosen based on the NaCl levels of four products to be used in the inoculated challenge studies and shelf-life studies, which had a range of NaCl concentrations of 1.85 to 3.6%. Cell cultures were resuscitated from glycerol stocks as described previously. Stationary-phase cells (500 l) were inoculated into fresh TSB (9.5 ml) amended with NaCl (no NaCl added [0.5% NaCl, wt/vol], 1.85% [wt/vol] NaCl, and 3.6% [wt/vol] NaCl, all ph 6.3 adjusted with HCl) to obtain cell levels of approximately 10 8 CFU/ml. Samples were transferred from these three cell suspensions (0.5 ml) to 50 ml of TSB, amended as above, to obtain cell suspensions with levels of approximately 10 6 CFU/ml. Sample tubes were prepared as described above. The cells were treated at 600 MPa for 0, 40, 60, 90, and 120 s. Pressure

3 J. Food Prot., Vol. 67, No. 8 HIGH-PRESSURE PROCESSING OF READY-TO-EAT MEATS 1711 treatment and subsequent enumeration were conducted as described previously, with two replicates. RTE meat products used in inoculated pack studies and shelf-life studies. In this study, four refrigerated, RTE, sliced, commercial meat products were used, as nominated by two Australian manufacturers of this category of products: Strassburg beef, low-fat pastrami, export sausage, and Cajun beef. The Strassburg beef is a cooked, cured, comminuted beef product (typical moisture, 66%; NaCl, 2%; sodium nitrite/nitrate, 63 ppm); the low-fat pastrami product is a cooked, cured, whole beef muscle product (typical moisture, 73%; NaCl, 1.85%; sodium nitrite/nitrate, 33 ppm); the export sausage is a cooked, cured, comminuted beef product produced entirely for export to the Asian market (typical moisture, 52%; NaCl, 1.85%; sodium nitrite/nitrate, 73 ppm); and the Cajun beef is a cooked, uncured, whole beef muscle encrusted with spices (typical moisture, 73%; NaCl, 3.6%; sodium nitrite/ nitrate, 57 ppm). These products where chosen based on (i) their high retail value; (ii) their potential to support L. monocytogenes growth over extended refrigerated storage; (iii) their ability to cover a range of cured and uncured and comminuted and whole muscle products; and/or (iv) their sale in the domestic or export market. Inoculated pack study. Cultures of L. monocytogenes 2472, 2542, 2345, 2343, or 2655 were prepared from glycerol stocks as described previously. Each strain (100 l) was added to the same 9.5 ml of 0.1% peptone to obtain a cocktail with approximately 10 8 CFU/ml. The cocktail was further diluted in 0.1% peptone so that 10 l contained approximately 10 4 CFU/ml. Low-fat pastrami, Strassburg beef, export sausage, and Cajun beef (approximately 25 g) were placed in oxygen barrier bags (Cryovac; oxygen transmission rate, 5 cc/m 2 /24 h/atm at 23 C, 75% relative humidity), and weights were recorded on the bags. The meats were inoculated with approximately 250 l of the L. monocytogenes cocktail (10 l/g of meat) to achieve a final concentration of approximately 10 4 CFU/g. The inoculum was spread over the surface of the meat, and the samples were massaged by hand for 30 s. The bags were vacuum packaged (Multivac, Sepp Haggenmuller GmbH and Co, Wolfeitschwenden, Germany), and the meats were pressure treated at 600 MPa, 20 C, for 180 s as described previously. of all products was conducted on the same day. The meats were stored at 4 C until needed. Microbiological testing was on days 1, 3, 14, 28, 42, 70, and 91 of storage post. Detection of L. monocytogenes during inoculated pack study. Both enumeration and enrichment for L. monocytogenes were conducted on (i) uninoculated, untreated meat samples, (ii) inoculated, untreated meat samples, and (iii) inoculated, pressuretreated meat samples after 1 day of storage at 4 C post pressure treatment. Subsequent sampling during the 91-day storage period was conducted on inoculated, pressure-treated meat samples only. Duplicate sample packages for each meat type were aseptically opened and were diluted (1:10, wt/wt) using sterile 0.1% peptone diluent. The packages were heat sealed and stomached for 2 min (Colworth 400, Seward, London, England). The samples (1 ml) were spread over three plates each of Oxford agar (Oxoid) and TSYEA. If necessary, serial dilutions were performed with 0.1% peptone diluent and plated onto Oxford agar and TSYEA. The plates were incubated at 37 C for 48 h. Duplicate samples were analyzed at each time interval. For selective enrichment, the sample packages were sprayed with 1% sodium hypochlorite, held for 10 min, then dried. The packages were aseptically opened, and sterile, half-strength Fraser broth (Oxoid) was added directly into the package to give a 1:10 dilution. The packages were heat sealed, hand massaged for 10 s, and incubated at 30 C for 24 h. The packages were opened, and samples (100 l) were transferred to sterile Fraser broth (10 ml) and incubated for 48 h at 37 C. One loopful of broth from both the packages containing the samples with half-strength Fraser and Fraser broth were streaked onto Oxford agar and incubated for 48 hat37 C. Any colonies that appeared on the plates were confirmed as L. monocytogenes by assessing colony morphology on TSYEA, Gram stain, catalase production, and the CAMP test. Shelf-life study of pressure-treated products. Four types of refrigerated RTE meat products (Cajun beef, export sausage, Strassburg beef, and low-fat pastrami) were obtained from two Australian manufacturers from their typical manufacturing line. All of the meats were received presliced. The low-fat pastrami and Strassburg beef products were in retail packs of 100 and 125 g and were held at 4 C before processing. Due to manufacturing schedules, the export sausage (received frozen) and Cajun beef were received in 1-kg bulk packs and were held at 20 C for 18 days before the study was initiated. Before, the export sausage and Cajun beef were thawed at 4 C, distributed into 500-g lots (for sensory testing) and 200-g lots (for chemical and microbiological analyses) in oxygen barrier packaging (Cryovac), and vacuum packaged using a Web-o-matic Easypack system (Maschinefabrick, Bochum, Germany). The low-fat pastrami and Strassburg beef were kept in their original retail vacuum packs. All products were pressure treated on the same day. The products were pressure treated at 600 MPa for 180 s at ambient temperature (approximately 20 C) using a 35-liter high-pressure unit (Avure Technologies, Inc.) in the Food Science Australia pilot plant located in Melbourne. Immediately after pressure treatment, the samples were held at 4 C and then shipped via refrigerated truck to Food Science Australia s Sydney facility for analyses during the shelf-life period. All products were stored at 4 C until required. The initial microbiological testing was conducted 4 days post pressure treatment, with sensory testing conducted 7 days post pressure treatment, after samples were confirmed to be microbiologically safe to consume. Subsequent microbial analyses were performed on days 11, 46, 74, and 95 (pressure-treated samples only). Sensory analyses followed 3 days later on days 14, 49, 77, and 98 (pressure-treated samples only). Microbiological analyses of the products in the shelf-life study. For microbial counts and enrichments, one package of each sample was removed from storage at 4 C. The package was sprayed with 70% ethanol and cut open using sterile scissors. Sterile tongs were used to remove one or two slices of meat (approximately 20 to 30 g) and placed in a stomacher bag (Biocorp, Melbourne, Australia). Sterile 0.1% peptone diluent was added to achieve a 10-fold dilution (wt/wt). The bag was heat sealed and stomached for 2 min. Samples (1 ml) were aseptically removed from the stomacher bags and were spread equally over three plates of each type of media required (Table 1). Microbiological testing was conducted to determine total aerobic plate count, total anaerobic plate count, and the presence of lactobacilli, Listeria spp., staphylococci, coliforms, Brochothrix thermospacta, and yeast and molds. The plates (Oxoid media) were incubated as described in Table 1 and counted. All product samples were tested in duplicate, except for the first and second (days 4 and 11) time points, from which microbiological analyses were conducted on only one sample of each product. Microbiological enrichments were conducted to detect coliforms, Salmonella spp., and Listeria spp. from the same samples used for plate counts. For coliforms enrichment, 100 l of sample

4 1712 HAYMAN ET AL. J. Food Prot., Vol. 67, No. 8 TABLE 1. Media and incubation conditions used to enumerate bacterial populations on low-fat pastrami, Strassburg beef, export sausage, and Cajun beef during the shelf-life study Microbial test Plate count media Incubation Total plate count Lactobacilli Listeria Staphylococci Coliforms Anaerobic plate count Brochothrix thermosphacta Yeasts and molds a In anaerobe jar (Oxoid). Standard plate count agar deman Rogosa Sharpe agar Listeria selective agar Baird-Parker agar Eosin methylene blue agar Brain heart infusion agar Streptomycin sulfate thallous acetate actidione agar Dichloran rose-bengal chloramphenicol agar 25 C/96 h 30 C/48 h 37 C/48 h 37 C/48 h 37 C/24 h 30 C/72 h a 30 C/48 h 25 C/5 days was added to lauryl tryptose broth (10 ml, Oxoid) and incubated at 37 C for 48 h. Samples from tubes with gas production (visualized by surfacing of Durham tube) were streaked onto eosin methylene blue agar. For Salmonella enrichment, 1 ml of sample was added to buffered peptone water (9 ml, Oxoid) and incubated at 37 C for 16 h. Subsequently, 100 l was transferred into Rappaport-Vassiliadis broth (10 ml, Oxoid) and incubated at 42 C for 24 h, after which the Rappaport-Vassiliadis broth was streaked onto xylose lysine desoxycholate agar (Oxoid). Listeria enrichment was conducted as described in the challenge studies section. Confirmatory tests were not necessary, because none of the samples tested during the study were presumptive positive. Chemical analyses of the products in the shelf-life study. The water activity (a w ) and ph of the products were monitored during the shelf-life study. The a w was determined using an Aqualab CX-3 a w meter (Graintec, Toowoomba, Australia). The ph was measured with a Beckman Coulter ph meter (model 390, Fullerton, Calif.), using a surface probe. Duplicate samples were tested. FIGURE 1. Listeria monocytogenes strains 2472 ( ), 2542 ( ), 2655 ( ), 2657 ( ), 2340 ( ), 2341 (#), 2342 ( ), 2343 ( ), and 2345 ( ) pressure treated in tryptic soy broth (ph 6.6, adjusted with 1 M HCl) at 600 MPa, 20 C. There were two replicates for each curve. The limit of detection was 10 CFU/ml. Consumer acceptance evaluation of the products in the shelf-life study. Consumer acceptance testing was conducted during the 98-day storage time (4 C), using approximately 40 consumers who currently consume cold, RTE meat products. All sensory testing was conducted in the sensory laboratory at Food Science Australia s Sydney facility according to International Standards on Sensory Analysis (2). Consumer acceptability of each sample was measured by hedonic appraisal of appearance, odor (aroma), flavor, texture, aftertaste, and overall liking, using a ninepoint hedonic scale (21). Additionally, consumers were asked to indicate the likelihood of sample purchase (given the right price), using a five-point purchase intent scale (21). Hedonic sensory data were analyzed using one-way analysis of variance (ANOVA) and Duncan s multiple comparison tests for means separation to determine differences (P 0.05) between hedonic ratings for the untreated (day 7) and pressure-treated meat samples at the evaluated time points (day 7, 14, 49, 77, or 98). Likewise, one-way ANOVA and Duncan s multiple comparison tests were used to determine differences (P 0.05) between hedonic ratings for the untreated and pressure-treated meat samples over the evaluated storage period. Purchase intent data were analyzed using the chisquare statistic to determine differences (P 0.05) between purchase intent ratings for the untreated (day 7) and pressure-treated meat samples at the evaluated time points. Differences (P 0.05) in purchase interest were further analyzed using one-way ANOVA and Duncan s multiple comparison tests for means separation. Likewise, chi-square, one-way ANOVA, and Duncan s multiple comparison tests were used to determine differences (P 0.05) between purchase intent ratings for the untreated and pressuretreated meat samples during the storage period. RESULTS Screening of L. monocytogenes strains for relative pressure resistance. The nine L. monocytogenes strains pressurized at 600 MPa (20 C) varied widely in their sensitivity to pressure (Fig. 1). For example, after 60 s of processing, between 1.2-log to more than 5.0-log reductions in cell numbers were achieved, depending on the strain. Strain 2542 was the most resistant, with a 1.2-log reduction achieved after 60 s at 600 MPa followed in relative resistance by strain 2655 (2.0-log reduction), strain 2345 (2.83- log reduction), and strain 2343 (3.23-log reduction). Strains 2340, 2342, 2657, 2341, and 2472 were the most sensitive, with 60 s at 600 MPa, 20 C, resulting in a greater than 5- log reduction in cell numbers observed. Determination of effect of NaCl on pressure inactivation of L. monocytogenes Because 2542 was the most resistant strain of those examined in this study, it was selected to investigate the effect of NaCl concentration on pressure inactivation. Due to the low level of inactivation of this strain after 60 s at 600 MPa, 20 C, longer processing

5 J. Food Prot., Vol. 67, No. 8 HIGH-PRESSURE PROCESSING OF READY-TO-EAT MEATS 1713 FIGURE 2. Listeria monocytogenes strain 2542 pressure treated at 600 MPa, 20 C, in tryptic soy broth (ph 6.3, adjusted with 1 M HCl) with 0.5% ( ), 1.85% ( ), and 3.6% ( ) (wt/vol) NaCl. There were two replicates for each curve. The limit of detection was 10 CFU/ml. times were selected. Inactivation of L. monocytogenes 2542 at 600 MPa, 20 C, was influenced by the NaCl level of the medium (Fig. 2). in TSB with no added NaCl (0.5% NaCl) resulted in the fastest inactivation, with a more than 5-log reduction achieved after 90 s at 600 MPa. As the NaCl levels increased, the level of inactivation decreased. For example, when cells were pressure treated in TSB with 1.85% NaCl at 600 MPa, 20 C, for 90 s, approximately a 3.5-log reduction was achieved. When the NaCl concentration was further increased to 3.6%, only a 2.5-log reduction was observed after pressure treatment for 90 s at 600 MPa, 20 C. Inoculated pack studies. On the initial day of testing, the L. monocytogenes counts on the inoculated, untreated meat samples were CFU/g for low-fat pastrami, CFU/g for Strassburg beef, CFU/g for export sausage, and CFU/g for Cajun beef, and all were positive for L. monocytogenes by selective enrichment. L. monocytogenes was not detected in any of the uninoculated, untreated meat samples by either enumeration or selective enrichment procedures. The ph measurements of the uninoculated, untreated meat samples ranged from 6.10 to 6.34, and a w ranged from to Selective enrichment of inoculated samples (approximately 25 g) treated at 600 MPa, 20 C for 180 s resulted in no L. monocytogenes positive samples 24 h after processing (Table 2). After 3 days of storage at 4 C, one lowfat pastrami and one Strassburg beef sample tested positive, via enrichment technique, for L. monocytogenes. Over the storage period of 13 weeks at 4 C, the Cajun beef samples never had a positive result, the low-fat pastrami had one additionally positive result at week 6 (one of two samples), the export sausage had positive results (one of two samples) at weeks 4, 10, and 13, whereas the Strassburg beef had positive results from day 3 to week 6, with no positive results at week 10 or 13 (Table 2). Enumeration of L. monocytogenes from meat samples pressure treated at 600 MPa, 20 C, for 180s on both nonselective (TSYEA) and selective (Oxford agar) media showed that all samples tested during a 10-week storage period had levels below the limit of detection ( 10 CFU/g). Although the Cajun beef did not have a positive result from the selective enrichment procedure throughout the 13-week storage study, on week 13, the Cajun beef sample had colonies present on both TSYEA and Oxford agar, for one replicate sample; however, high numbers and confluence of colonies on the plate made it difficult to be confident that the cell morphology was consistent with that typical for L. monocytogenes. The Strassburg beef and the export sausage also had viable counts of L. monocytogenes on one replicate sample each on week 13, which is consistent with the sporadic positives seen with the enrichment method used throughout the storage period. Additionally, the Strassburg beef sample also had significant background flora present on the TSYEA plate, at levels approximately twofold higher than those of the colonies identified as L. monocytogenes. Microbiological analyses of the products in the shelf-life study. All pressure-treated low-fat pastrami, Strassburg beef, export sausage, and Cajun beef were microbiologically safe for consumer testing throughout the 98 days of refrigerated storage. There were no samples positive (via enrichment techniques) for Listeria spp., Salmonella spp., or coliforms in the untreated (tested on day 4 only) or pressure-treated samples at any time. The untreated meat samples had levels of aerobic bacteria, Listeria spp., staphylococci, coliforms, and Brochothrix thermosphacta below the detection limit ( 10 CFU/g) in all four products. Low levels ( 500 CFU/g) of lactobacilli, anaerobic bac- TABLE 2. Pressure-treated (600 MPa, 20 C, 180 s) low-fat pastrami, Strassburg beef, export sausage, and Cajun beef samples positive ( ) or negative ( ) for Listeria monocytogenes (by selective enrichment, duplicate samples) over 91 days of storage at 4 C a Results on day: Product Low-fat pastrami Strassburg beef Export sausage Cajun beef a Initial L. monocytogenes count before high-pressure processing () was approximately 10 4 CFU/g; sample weight was approximately 25 g. Limit of detection was 0.04 CFU/g.

6 1714 HAYMAN ET AL. J. Food Prot., Vol. 67, No. 8 teria, and yeast and molds (mainly yeast) were detected in the untreated low-fat pastrami and Cajun beef samples. Aerobic bacteria, lactobacilli, Listeria spp., staphylococci, coliforms, anaerobic bacteria, B. thermosphacta, and yeast and molds in the low-fat pastrami samples were at undetectable levels ( 10 CFU/g) 4 days post pressure treatment and remained at undetectable for 95 days post pressure treatment. The only exceptions were on day 11, where a low level of aerobic bacteria was detected ( 200 CFU/g), and on day 95, where low levels of anaerobic bacteria were detected ( 100 CFU/g). Aerobic bacteria, lactobacilli, Listeria spp., staphylococci, coliforms, anaerobic bacteria, B. thermosphacta, and yeast and molds in the Strassburg beef samples were at undetectable levels ( 10 CFU/g) 4 days post pressure treatment processing and remained at undetectable levels for 95 days post pressure treatment. The only exception was on day 11, where a low level of aerobic bacteria was detected ( 10 3 CFU/g). Aerobic bacteria, lactobacilli, Listeria spp., staphylococci, coliforms, anaerobic bacteria, B. thermosphacta, and yeast and molds in the export sausage samples were at undetectable levels ( 10 CFU/g) 4 days post pressure treatment and remained at undetectable levels for 95 days post pressure treatment. The two exceptions occurred on day 11, where a low level of yeast and molds was detected ( 100 CFU/g), and on day 46, where a low level of aerobic bacteria was detected ( 100 CFU/g). Aerobic bacteria, lactobacilli, Listeria spp., staphylococci, coliforms, anaerobic bacteria, B. thermosphacta, and yeast and molds in the Cajun beef samples were at undetectable levels ( 10 CFU/g) 4 days post pressure treatment and remained at undetectable levels for 95 days post pressure treatment. On day 46, low levels of aerobic bacteria, lactobacilli, staphylococci, and anaerobic bacteria were detected ( 10 3 CFU/g), and on day 95, low levels of lactobacilli and yeasts and molds were detected ( 100 CFU/g). Chemical analyses of the products during the shelflife study. The ph and a w of the four products in the trial were measured for the untreated samples and were measured over the chilled storage of the pressure-treated samples. The ph of the untreated samples had a range in ph of 5.89 to did not affect the ph of these products, and the ph did not change over 95 days of storage at 4 C (data not shown). The a w of the untreated samples had a range of to did not affect the a w of the products nor did the a w change over 95 days of storage at 4 C (data not shown). Consumer acceptability evaluation of the products during the shelf-life study. Sensory testing on day 7 post pressure treatment was performed on the untreated and pressure-treated low-fat pastrami, Strassburg beef, export sausage, and Cajun beef meat samples. No differences (df 1, 78; P 0.05) were observed between the untreated and pressure-treated meat samples for acceptability or purchase intent at day 7 of refrigerated storage. Likewise, sensory testing on day 14 was performed on the pressure-treated meat samples, and the results were compared with consumer acceptability ratings for the untreated meat samples (day 7). No differences (df 1, 72; P 0.05) in consumer acceptability and purchase intent ratings were observed between the untreated (day 7) and corresponding meat samples at day 14 of refrigerated storage. Export sausage was excluded from consumer evaluation on day 49 due to gray discoloration. Discoloration was thought to be due to residual oxygen surrounding the sample after vacuum packaging, because discoloration was only evident on the visually exposed parts of the sample. On day 49, there were some differences in consumer acceptability ratings between the untreated (day 7) and corresponding pressure-treated meat samples (Table 3). Consumers rated the aftertaste of the pressure-treated low-fat pastrami and Cajun beef meat samples higher (df 1, 74; P 0.05) on day 49 of refrigerated storage compared with the corresponding untreated meat samples on day 7 of refrigerated storage. Furthermore, overall liking of the pressure-treated Cajun beef samples received a higher (df 1, 74; P 0.05) rating on day 49 of refrigerated storage compared with the untreated Cajun beef sample on day 7 of refrigerated storage. On day 77 of refrigerated storage, there was some gray discoloration in export sausage and, to a lesser extent, in the Cajun beef samples. Because the discoloration was due to vacuum packaging failure, not, the most discolored portions of each sample were separated and discarded, whereas the pinker portions were presented to consumers for acceptability evaluation. There were several differences (df 1, 71; P 0.05) in acceptability ratings between the untreated (day 7) and pressure-treated samples on day 77 of refrigerated storage (Table 4). The flavor of the pressuretreated low-fat pastrami sample received a higher (df 1, 71; P 0.05) rating on day 77 of refrigerated storage compared with the untreated meat sample (day 7). The flavor, aftertaste, and overall liking of the pressure-treated Cajun beef sample received higher (df 1, 71; P 0.05) ratings on day 77 of refrigerated storage compared with the untreated Cajun beef sample (day 7). Furthermore, the chisquare statistic used to analyze differences in purchase intent ratings revealed that consumers were more willing to purchase the pressure-treated Cajun beef on day 77 of refrigerated storage compared with the untreated Cajun beef sample (day 7). On day 98 of refrigerated storage, there were no differences (df 1, 78; P 0.05) in consumer acceptability or purchase intent ratings between the pressure-treated and untreated (day 7) meat samples. In summary, comparison of consumer hedonic ratings for the untreated (day 7) and corresponding pressure-treated meat samples during the evaluated storage period revealed no deterioration (df 5, 217; P 0.05) in the sensory quality of the Strassburg beef, export sausage (df 4, 182; P 0.05), low-fat pastrami, and Cajun beef meat samples during the 98-day evaluation period. DISCUSSION The nine L. monocytogenes strains examined in this study varied in their resistance to, with a 1.2-log to a

7 J. Food Prot., Vol. 67, No. 8 HIGH-PRESSURE PROCESSING OF READY-TO-EAT MEATS 1715 TABLE 3. consumer acceptability scores for the untreated (day 7) and high-pressure processed () ready-to-eat meat samples on day 49 of storage at 4 C a Product Appearance Aroma Flavor Texture Aftertaste Overall liking Purchase intent Low-fat pastrami Strassburg beef Cajun beef a All scores are based on a nine-point hedonic scale, except purchase intent, which is based on a five-point scale. more than 5-log reduction after 60 s at 600 MPa, 20 C. This agrees with previous publications in which strain variability has been observed (1, 16, 24, 26). The five most resistant strains of those examined were 2542 (most resistant), 2472, 2345, 2343, and 2655, and therefore these strains were used as a cocktail for the inoculated challenge tests. Because the products used in this study had various NaCl concentrations (1.85 to 3.6%), the effect of NaCl concentration on pressure inactivation was examined. Since L. monocytogenes 2542 was the most resistant of the nine strains examined, it was selected for these inactivation kinetic studies. The pressure inactivation kinetics studies were different when cells were treated in TSB with varying salt concentrations. As the level of salt was increased from 0.5 to 3.6% (highest NaCl concentration in the four meat products used in the challenge studies), the level of inactivation decreased. These results indicate that NaCl may afford some protection to L. monocytogenes during, perhaps via stabilization of critical proteins in the cell membrane. The results from inactivation kinetics studies, particularly those with strain 2542 when pressure treated in TSB with 3.6% NaCl was used, indicated that processing times at 600 MPa, 20 C, would need to be greater than 120 s to achieve a 4-log CFU/ml reduction of L. monocytogenes. Additionally, preliminary inoculated challenge tests (data not shown) indicated that processing inoculated meat samples (initial inoculum levels of 10 4 CFU/g) for 180 s at 600 MPa, 20 C, would allow for no detectable levels of L. monocytogenes immediately after processing, using enrichment techniques. For these reasons, pressure treatment at 600 MPa, 20 C, for 180 s was the process chosen for both the inoculated challenge studies and the shelf-life studies. The inoculated challenge studies showed that levels of L. monocytogenes would remain below detectable levels during at least a 10-week storage period at 4 C for some products, but other products had sporadic positive results from selective enrichment procedures and some packages had countable levels of L. monocytogenes at 13 weeks post pressure treatment. These results indicate the possibility of recovery and growth of L. monocytogenes cells during 13 weeks of storage at 4 C. This finding would need to be confirmed with further studies. Additionally, the results indicated that a slightly longer processing time or higher pressure may be necessary to ensure nondetectable levels if the initial level of L. monocytogenes on product is assumed to be 10 4 CFU/g. However, a recently published survey of L. monocyto-

8 1716 HAYMAN ET AL. J. Food Prot., Vol. 67, No. 8 TABLE 4. consumer acceptability scores for the untreated (day 7) and high-pressure processed () ready-to-eat meat samples on day 77 of storage a Product Appearance Aroma Flavor Texture Aftertaste Overall liking Purchase intent Low-fat pastrami Strassburg beef Export sausage Cajun beef a All scores are based on a nine-point hedonic scale, except purchase intent, which is based on a five-point scale. genes in RTE foods indicated that levels of L. monocytogenes in luncheon meats are typically lower than 10 4 CFU/g (8). RTE luncheon meats were one of eight categories of foods examined during a 23-month period. Of the 9,199 presliced luncheon meat (ham [pork or poultry], bologna [pork, beef, turkey, or a mixture of these], poultry [turkey or chicken; smoked or not smoked]) samples examined, only 82 (0.89%) tested positive for L. monocytogenes. Most samples had levels of less than 10 2 CFU/g, with only one sample having initial levels of 10 3 to 10 4 CFU/g. This indicated that most retail RTE meats would have levels much lower than the level chosen for these challenge studies, indicating that pressure treatment of 600 MPa for 180 s could be used successfully as the final in-package pasteurization step in the commercial production of refrigerated, RTE meats. Further evaluation of the effects of various components in the meat products, such as fat level, spices, and acids, on the effectiveness of, would aid in the development of commercial in-package pasteurization processes, which would ensure the safety and extend the shelf life of refrigerated, RTE meat products. The microbiological analyses of RTE meat products used in the shelf-life study (low-fat pastrami, Strassburg beef, export sausage, and Cajun beef) showed that (600 MPa, 20 C, 180 s) was effective in keeping levels of aerobic bacteria, lactobacilli, Listeria spp., staphylococci, coliforms, anaerobic bacteria, B. thermosphacta, and yeast and molds to below the detectable limits ( 10 CFU/g) or

9 J. Food Prot., Vol. 67, No. 8 HIGH-PRESSURE PROCESSING OF READY-TO-EAT MEATS 1717 at low levels throughout the 95 days of storage at 4 C. Additionally, at no time during the shelf-life trial did any samples test positive (via enrichment methods) for L. monocytogenes, coliforms, or Salmonella spp. These findings are in agreement with Lopez-Carballo et al. (17), who reported that at 400 MPa, 7 C, for 20 min significantly reduced numbers of B. thermosphacta, coliforms, and Baird-Parker flora in sliced ham and that these microorganisms were not detected over 35 days of refrigerated storage. These results indicate that can successfully be used as an in-package pasteurization method to significantly extend the refrigerated shelf life of RTE meat products with regard to microbial safety and stability. Results from the consumer trial revealed that was able to maintain the sensory quality of all four RTE meats during a 98-day period of refrigerated storage. The hedonic ratings provided by untrained consumers revealed that there was no deterioration in the integrated sensory quality of the meat samples throughout the evaluation period. Although it is noteworthy that average consumer ratings for pressuretreated samples were, on occasion, higher than the untreated samples, it is unlikely that contributed to the apparent enhancement of sensory character during storage; however, conclusive elucidation of such an effect would require more comparative-type studies, such as sensory profiling in conjunction with electronic nose analysis or separative volatile measurement techniques. Notwithstanding, the results demonstrated the ability of to maintain the sensory quality of Strassburg beef, low-fat pastrami, export sausage, and Cajun beef samples for an extended period of refrigerated storage (98 days). Furthermore, sensory testing was in line with minimal microbial growth and no change in chemical stability during a similar storage period. The results from this study show that (as an inpackage nonthermal pasteurization method) can be a powerful intervention strategy for controlling L. monocytogenes in RTE refrigerated meats as part of a good overall hazard analysis critical control point (HACCP) program. The U.S. Department of Agriculture Food Safety Inspection Service has listed as a potential postlethality treatment, which may be used to reduce or eliminate L. monocytogenes in RTE meat and poultry products if included in the HACCP plan and the effectiveness is validated (29). Although the inoculated challenge testing showed sporadic positives for the presence of L. monocytogenes, a greater than 4-log CFU/g reduction was achieved, as indicated by enumeration methods during at least 10 weeks of storage at 4 C and enrichment techniques performed immediately post processing. The shelf-life studies, which included both microbiological analyses and consumer acceptance testing, indicated that the refrigerated shelf life of commercially available RTE sliced meat products could be greatly extended from their current refrigerated shelf life (approximately 45 to 50 days) to at least 98 days while maintaining the eating quality and microbiological safety and quality. ACKNOWLEDGMENT This project was cofunded by Meat and Livestock Australia, Ltd., project PRMS.033. REFERENCES 1. Alpas, H., F. Kalchayanand, F. Bozoglu, A. Sikes, C. P. Dunne, and B. Ray Variation in resistance to hydrostatic pressure among strains of foodborne pathogens. Appl. Environ. Microbiol. 65: Anonymous ISO 6658: sensory analysis, methodology, general guidance. International Organisation for Standardisation, Paris, France. 3. Farber, J. M., and E. Daley Presence and growth of Listeria monocytogenes in naturally contaminated meats. Int. J. 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