Lactic Acid Sprays Reduce Bacterial Pathogens on Cold Beef Carcass Surfaces and in Subsequently Produced Ground Beef

58 Journal of Food Protection, Vol. 64, No. 1, 2001, Pages 58 62 Copyright q, International Association for Food Protection Lactic Acid Sprays Reduce Bacterial Pathogens on Cold Beef Carcass Surfaces and in Subsequently Produced Ground Beef A. CASTILLO, L. M. LUCIA, D. B. ROBERSON, T. H. STEVENSON, I. MERCADO, AND G. R. ACUFF* Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA MS 00-192: Received 9 June 2000/Accepted 3 August 2000 ABSTRACT Organic acids have been shown to be effective in reducing the presence of pathogenic bacteria on hot beef carcass surfaces; however, application for decontaminating chilled carcasses has not been fully evaluated. In this study, a postchill, 30-s lactic acid spray (500 ml of 4% L-lactic acid, 558C) was applied onto outside rounds that had been contaminated with Escherichia coli O157:H7 and Salmonella Typhimurium, subsequent to prechill hot carcass treatments consisting of water wash alone or water wash followed by a 15-s lactic acid spray (250 ml of 2% L-lactic acid, 558C). The prechill treatments reduced both pathogens by 3.3 to 3.4 log cycles (water wash alone) to 5.2 log cycles (water wash and lactic acid). In all cases, the postchill acid treatment produced an additional reduction in E. coli O157:H7 of 2.0 to 2.4 log cycles and of 1.6 to 1.9 log cycles for Salmonella Typhimurium. The counts of both pathogens remained signi cantly lower in ground beef produced from the outside rounds that received prechill and postchill acid spray than from those that received a postchill spray only. These data indicate that organic acid sprays may be successfully applied for pathogen reduction in beef carcass processing after the cooler, especially when combined with prechill treatments. Organic acid rinses have been proposed as effective, inexpensive carcass interventions (2, 4 6, 10, 11, 13, 18, 21, 25), with lactic or acetic acid being the most commonly used compound in beef carcass decontamination. Their ability to reduce pathogens or other organisms of fecal origin on beef surfaces has been extensively studied (14, 18, 23, 27). A continued antimicrobial effect has also been observed during storage of meat after spraying lactic or acetic acid solutions on hot carcass surfaces (14, 15, 20, 24). A concern related to spraying beef carcasses with organic acid is the reported resistance of E. coli O157:H7 to low ph (8, 22, 26). However, recent studies indicate that lactic or acetic acid sprays, when applied at 558C, are effective at reducing levels of Salmonella or E. coli O157:H7 (7, 18). In different studies, the temperature of the acid solution has been found to have a profound effect on the magnitude of reductions in bacterial counts on carcass surfaces (2, 3, 17). Therefore, the effectiveness of these interventions may vary, depending on whether they are applied onto hot or cold carcass surfaces. Although organic acid sprays have been shown to produce large bacterial reductions on the surface of hot beef carcasses (10), other reports on decontamination of cold, fabricated beef indicated that organic acid sprays may not be as effective when applied to cold cuts (1, 9, 12). Nevertheless, the use of organic acid sprays for reducing pathogenic bacteria on cold carcasses has not been fully studied, and its usefulness as an after-the-cooler intervention remains unclear. Effective application of an organic acid treatment to chilled carcass surfaces would allow * Author for correspondence. Tel: 979-845-4425; Fax: 979-862-3475; E-mail: gacuff@tamu.edu. Technical article from the Texas Agricultural Experiment Station. implementation of a decontamination intervention to supplement carcass decontamination during slaughter, possibly assisting in preserving the safety of fabricated or ground meat should recontamination occur after chilling. The objective of this study was to develop a lactic acid spray suitable for treating chilled beef carcass surfaces to study its effectiveness in reducing populations of E. coli O157:H7 and Salmonella Typhimurium and to determine the potential for continued antimicrobial effect of lactic acid in ground beef prepared from sprayed cold outside rounds. MATERIALS AND METHODS Microorganisms and inoculum preparation. Rifampicinresistant E. coli O157:H7 (derived from an isolate from ground beef implicated in a foodborne disease outbreak) and Salmonella enterica serovar Typhimurium (derived from Salmonella Typhimurium ATCC 13311) were grown on tryptic soy agar (Difco Laboratories, Detroit, Mich.) slants and stored at room temperature. Growth curves, heat resistance, and acid sensitivity of the mutant strains were determined to be virtually indistinguishable from the parent strain (18, 19). Three days before each experiment, these microorganisms were resuscitated by two consecutive transfers to tryptic soy broth (Difco), incubating at 358C for 18 to 24 h. The day before the experiment, the two microorganisms were grown separately at 358C in 500 ml of tryptic soy broth. A 12-h culture of each microorganism was dispensed in sterile centrifuge tubes (30 ml) and harvested by centrifugation at 15,000 rpm for 15 min at 48C. The pellet for each microorganism then was resuspended in 5 ml of sterile peptone (Difco) water. Several bags with inoculum were prepared by mixing the whole contents of a tube containing each bacterial pathogen with 10 g of fresh bovine feces inside a sterile stomacher bag. By this procedure, each inoculum bag was expected to contain approximately 10 10 cells of each marker pathogen. A portion of the inoculated fecal

J. Food Prot., Vol. 64, No. 1 DECONTAMINATION OF CHILLED BEEF CARCASSES 59 suspension was tested to con rm the number of marker organisms per g by plating onto lactose-sul te-phenol red-rifampicin(lspr) agar, incubating at 358C for 24 h. LSPR agar was prepared by adding yeast extract, beef extract, lactose, sodium sul te, ferrous sulfate, phenol red, cycloheximide, and rifampicin to previously dissolved tryptic soy agar, according to the formulation proposed by Castillo et al. (6). Rifampicin-resistant E. coli produce yellow colonies on the medium, whereas rifampicin-resistant Salmonella develop colonies with a black center surrounded by a pink halo. Carcass selection. Fed steers or heifers typical of those entering the U.S. meat supply were selected for use in the study. After transporting to the Texas A&M University Rosenthal Meat Science and Technology Center, cattle were slaughtered and dressed in the university abattoir following U.S. Department of Agriculture, Food Safety and Inspection Service regulated commercial procedures. The two outside round regions were separated from the remainder of the carcass just subsequent to carcass splitting. Carcasses were not washed or decontaminated in any manner before the outside rounds were obtained for use in this study. The outside rounds were transported from the slaughter oor in insulated containers to the laboratory located in an adjacent building, where they were contaminated with the prepared feces and subjected to the different treatments. Beef carcass surfaces and inoculation. A 400-cm 2 surface area (20 by 20 cm) was marked on the outer surface of each hotboned outside round. The pathogen-inoculated fecal suspension (10 g) then was removed from the sterile bag and spread with a spatula onto the marked area on the surface of the cuts by smearing in a one-way crossing motion. The spatula was cleaned and sterilized between inoculations by dipping into 70% alcohol followed by igniting with a ame. This approach produced initial counts of approximately 8.0 log 10 CFU/cm 2. Uninoculated feces were used to contaminate outside round surfaces during preliminary studies to select the best postchill treatment. Before and after inoculation, a 10-cm 2 sample was collected and tested for rifampicin-resistant E. coli and Salmonella by plating onto LSPR agar plates. To obtain the sample, a sterile borer was used to initially cut (about 2 to 3 mm deep) a 10-cm 2 area, followed by slicing the surface sample (taking 2 mm or less of interior tissue) from the carcass surface area using a sterile scalpel and forceps. Each sample was mixed with 100 ml of sterile diluent (0.1% peptone) in a stomacher bag and pummeled for 1 min in a Stomacher 400 before examination. Selection of a postchill lactic acid treatment. Outside rounds were cut in half, and the original carcass surface section of each half was contaminated by spreading uninoculated feces over 400 cm 2 as described above (n 5 16). Immediately after inoculation, all pieces were sampled and tested for numbers of E. coli by plating onto E. coli Petri lm and incubating for 24 h at 358C. The pieces then were washed using an automated carcass water wash consisting of an initial low-pressure manual wash followed by a high-pressure wash in an automated spray cabinet (Chad Co., Lenexa, Kan.). In the initial hand wash, the outside round to be treated was hung in the cabinet in the same orientation as it would be positioned on an intact carcass, and 1.5 liter of water (approximately 258C) was sprayed at 10 psi (69 kpa) for 90 s using a handheld, noncorrosive, polyethylene compressed-air sprayer (10.56 l, Universal-Gerwin, Saranac, Mich.). An automated high-pressure wash was then applied consisting of spraying 5 liters of potable water at 358C for 9 s, starting at an initial pressure of 250 psi for 4 s and gradually increasing to 400 psi within 2 s, maintaining this pressure for 3 s to complete a total treatment time of 9 s. The outside round halves then were sampled and tested for E. coli to measure the reduction in counts of this indicator by the automated water wash. After washing, the cuts were chilled at 48C for 24 h and then tested again for E. coli to determine a possible reduction caused by the chilling step. Duplicate pieces then were separated and assigned to one of the following postchill lactic acid treatments: (i) 2% L-lactic acid at 558C sprayed for 15 s, (ii) 2% L-lactic acid at 558C sprayed for 30 s, (iii) 2% L-lactic acid at 658C sprayed for 15 s, (iv) 2% L-lactic acid at 658C sprayed for 30 s, (v) 4% L-lactic acid at 558C sprayed for 15 s, (vi) 4% L-lactic acid at 558C sprayed for 30 s, (vii) 4% L-lactic acid at 658C sprayed for 15 s, and (viii) 4% L-lactic acid at 658C sprayed for 30 s. E. coli counts obtained before wash, after wash, and after chill were used as controls. The sprays were applied using a handheld, noncorrosive, polyethylene compressedair sprayer (10.56 l, Universal-Gerwin) with the nozzle calibrated to deliver 1,000 ml of solution per min. The treated cuts were sampled and tested for numbers of E. coli following the procedure described above. E. coli counts on postchill treated outside rounds halves were compared with counts obtained after inoculation, water wash, and chill to determine the reduction achieved by the postchill lactic acid sprays. Pathogen reduction by prechill and postchill lactic acid sprays. The original carcass surface sections of whole, hot-boned outside rounds (n 5 18) were inoculated with rifampicin-resistant E. coli O157:H7 and Salmonella Typhimurium contained in a fecal suspension, according to the procedure described above, and then separated in six groups of three (six repetitions). For each trial, each inoculated outside round was cleaned using an automated carcass water wash, and then the surface of one of the three rounds was sanitized by spraying 2% L-lactic acid at 558C for 15 s. One of the two rounds that were not sprayed was used as a nontreated control and the other for further treatment comparison. All rounds in each repetition were sampled and tested for numbers of rifampicin-resistant E. coli O157:H7 and Salmonella Typhimurium, plating appropriate dilutions of the initial suspension. Immediately after the corresponding prechill treatment, each carcass surface section was hung in a refrigerated cooler in the microbiology laboratory at 48C for 24 h and then examined again for levels of both marker organisms. Of the two carcass surface sections that were not applied a prechill lactic acid spray, one was left untreated after chilling to serve as an unsanitized control. The two remaining cuts were treated with a postchill sanitizing treatment consisting of spraying 4% L-lactic acid at 558C for 30 s, simulating a prefabrication decontamination step. All carcass surfaces were sampled again after postchill treatment and tested for numbers of both marker pathogens. The examination of the carcass surface sections was intended to determine the numbers of both pathogens and how the treatments affected these numbers. Sampling was achieved by excising a 10-cm 2 sample as described above, mixing it with 100 ml of sterile 0.1% peptone water in a stomacher bag, and pummeling for 1 min in a Stomacher 400. Numbers of rifampicin-resistante. coli O157:H7 and Salmonella Typhimurium were simultaneously determined from these samples by placing 1.0 ml (divided into 0.25-ml aliquots over four plates) and 0.1 ml of the beef-peptone homogenate and 0.1-ml volumes of appropriate decimal dilutions of the same on prepoured and dried LSPR agar plates. Plates were incubated at 378C for 24 h before counting and reporting log 10 numbers of rifampicin-resistant E. coli O157:H7 or Salmonella Typhimurium per g. The effectiveness of the prechill and postchill treatments was determined by comparing the counts of both path-

60 CASTILLO ET AL. J. Food Prot., Vol. 64, No. 1 TABLE 1. Reduction of E. coli by prechill water wash followed by different postchill lactic acid treatments Treatment Control b Water wash d Water wash then 24-h chill Postchill lactic acid treatment 2%, 558C, 15 s 2%, 558C, 30 s 2%, 658C, 15 s 2%, 658C, 30 s 4%, 558C, 15 s 4%, 558C, 30 s 4%, 658C, 15 s 4%, 658C, 30 s Log 10 CFU/cm 2 6 SEM 5.3 6 0.112 A c 2.4 6 0.186 B 1.7 6 0.197 BC 1.1 6 0.400 C 1.4 6 0.650 BC 1.2 6 0.550 BC Log reduction 6 SEM a 0.0 6 0.000 A 2.9 6 0.078 B 3.5 6 0.106 BC 4.2 6 0.600 CD.4.7 6 0.600 D 4.0 6 0.150 CD.4.4 6 0.400 D 4.2 6 0.250 D.4.8 6 0.550 D.4.5 6 0.300 D.4.6 6 0.350 D a Log reduction 5 (log 10 CFU/cm 2 after inoculation [control]) 2 (log 10 CFU/cm 2 after treatment). SEM, standard error of the mean. b Control: outside round after inoculation with 10 g of feces spread over approximately 400 cm 2. c Numbers within columns followed by same letter are not signi cantly different (P, 0.05). d Water: 1.5-liter hand wash (90 s, 10 psi) followed by 5-liter automated cabinet wash (9 s, 250 to 400 psi) at 358C. ogens on treated carcass surfaces with counts on control, nontreated carcass surfaces. Effect of lactic acid sprays on pathogen survival in ground beef. For three of the six repetitions of the above procedure, sterile meat grinders were used to grind the treated outside rounds. Each outside round was coarse ground using a 2-in. plate and then ne ground using a 3-in. plate before stuf ng into 2-lb oxygen-impermeable chubs. Chubs were stored at 48C, and three replicate chubs from each treatment were sampled at 0, 7, 14, and 21 days of storage. Sampling was achieved by aseptically removing 10 g using a sterile scalpel and forceps. The sample was placed in a sterile stomacher bag with 90 ml of sterile 0.1% peptone and pummeled for 1 min in a Stomacher 400. Appropriate dilutions of the resulting suspension were surface plated onto LSPR agar plates and incubated for 24 h at 358C before obtaining counts for each pathogen. The effect of the different treatments on the survival of both pathogens was determined by comparing counts. Data analysis. Microbiological count data were transformed into logarithms before obtaining means and performing statistical analyses. Analysis consisted of analysis of variance and general linear model procedures, followed by determination of signi - cance in the analysis of treatment effects using Duncan s multiple range test. RESULTS AND DISCUSSION Selection of a postchill lactic acid treatment. Studies conducted to select an effective postchill treatment of fecescontaminated outside rounds consisted of comparing E. coli counts on rounds to which sprays of solutions with different concentrations of lactic acid were applied. Data in Table 1 indicate that an automated water wash alone was capable of reducing E. coli counts by 2.9 log 10 CFU/cm 2 and that no further signi cant reduction was observed after chilling the washed carcass surfaces for 24 h at 48C. Similar reductions have been observed in previous studies, where an automated water wash was combined with subsequent organic acid or hot water sprays (6, 7, 18). When lactic acid sprays were applied on chilled rounds, sprays with 2% lactic acid produced additional reductions (P, 0.05) but detectable numbers of E. coli remained. However, 4% lactic acid at 558C sprayed for 30 s or 4% lactic acid at 658C sprayed either for 15 or 30 s consistently produced undetectable levels of E. coli. Based on these results, a 4% lactic acid solution at 558C sprayed for 30 s was chosen as the postchill treatment to be used in subsequent studies. Pathogen reduction by prechill and postchill lactic acid sprays. Data on the reduction of E. coli O157:H7 and Salmonella Typhimurium by different treatments are shown in Table 2. The numbers shown indicate the log reductions in the initial count of each pathogen due to water wash and the additional reduction obtained after each subsequent treatment. The control group of outside rounds received only an automated water wash. Depending on the pathogen, initial counts of 7.0 to 7.3 log 10 CFU/cm 2 were reduced by 2.4 to 2.6 log cycles by water wash, with a reduction of only 0.1 to 0.2 log 10 /cm 2 after chilling at 48C for 24 h. Similar results were obtained during preliminary studies with fecal E. coli (Table 1). A second group, named water wash, received a prechill water wash followed by a postchill lactic acid spray (4% L-lactic acid at 558C sprayed for 30 s). In this group there was a signi cant reduction (0.8 to 1.4 log cycles) for both pathogens after chilling. This behavior was inconsistent with all other experiments conducted during this study. When a postchill lactic acid spray was applied, an additional reduction of 1.9 to 2.4 log cycles was achieved. Adding the reductions obtained after water wash and after the postchill lactic treatment produced a total reduction of 5.3 to 5.7 log cycles. This reduction is similar to those reported by several workers for bacterial pathogens on beef carcass surfaces after a combined treatment consisting of water wash followed by an organic acid treatment (7, 11, 13, 14, 16). The last group, named water wash plus prechill lactic acid spray, included a conventional combined treatment of water wash followed by 2% L-lactic acid at 558C sprayed for 15 s and then a postchill treatment, which has been described above. The prechill treatment reduced the counts of both E. coli O157:H7 and Salmonella Typhimurium by 5.2 log cycles. Reductions of this magnitude have been consistently reported in previous studies (7, 18). However, additional reductions of 1.6 to 2.0 log 10 /cm 2 were obtained in this group of rounds after a postchill spray was applied. A total log reduction of 6.8 to 7.2 was achieved by applying combined, sequential treatments consisting of prechill and postchill lactic acid sprays. This reduction may be large enough to recommend a lactic acid treatment of cold carcasses before fabrication in meat plants, when the hot carcasses have received an appropriate decontamination treatment, as is the case currently in many beef slaughter plants.

J. Food Prot., Vol. 64, No. 1 DECONTAMINATION OF CHILLED BEEF CARCASSES 61 TABLE 2. Reduction of E. coli O157:H7 and Salmonella Typhimurium on inoculated outside round beef carcass surface regions after treatment with water wash combined with pre- and postchill lactic acid sprays a Log reduction after previous treatment 6 SEM b Prechill treatment Sampling before and after chilling E. coli O157:H7 Salmonella Typhimurium Control (n 5 4) After water wash After chilling d 2.4 6 0.266 B c 0.1 6 0.411 D 2.6 6 0.212 BC 0.2 6 0.268 DE Water wash (n 5 6) After water wash After chilling After postchill lactic acid spray 3.3 6 0.171 B 0.8 6 0.452 D 2.4 6 0.705 B 3.4 6 0.130 B 1.4 6 0.544 CD 1.9 6 0.738 C Water wash plus prechill lactic acid spray (n 5 6) After water wash plus prechill lactic acid spray After chilling After postchill lactic acid spray 5.2 6 0.280 A 0.0 6 0.529 D 2.0 6 0.514 BC 5.2 6 0.280 A 0.0 6 0.396 E 1.6 6 0.225 C a Water wash: 1.5-liter hand wash (90 s, 10 psi) followed by 5-liter automated cabinet wash (9 s, 250 to 400 psi) at 358C. Prechill lactic acid spray: 250 ml of 2% L-lactic acid sprayed at 10 psi for 15 s at 558C; postchill lactic acid spray: 500 ml of 4% L-lactic acid sprayed at 10 psi for 30 s at 558C. b Log reduction 5 (log 10 CFU/cm 2 after immediate previous treatment) 2 (log 10 CFU/cm 2 after treatment). SEM, standard error of the mean. c Numbers within columns with same letter are not signi cantly different (P, 0.05). d Chilling, inoculated outside rounds hung for 24 h at 48C. No reduction in counts of both pathogens was observed after chilling, which is consistent with all experiments in this study except for the water wash group, where a signi cant reduction was observed after the chilling step, as previously mentioned. Even though this may appear to be inconsistent, this observation was repeatedly observed. Additional research is, therefore, needed to determine if there is a difference in pathogen susceptibility to cold temperatures when a water wash is applied alone compared with water wash followed by a prechill lactic acid spray. Effect of lactic acid sprays on pathogen survival in ground beef. Counts of E. coli O157:H7 and Salmonella Typhimurium were obtained from chubs of ground beef produced from outside rounds of each treatment group. Counts of E. coli O157:H7 obtained from ground beef prepared from rounds for each of these treatment groups during 21 days of storage at 48C are shown in Figure 1. Counts remained signi cantly lower on every sampling day in ground beef made from rounds that had received a water wash followed by a prechill lactic acid spray and then a postchill lactic acid spray. Furthermore, the slope of the trend line shown for each data series indicates that there was a small but steady reduction in counts of ground beef produced from rounds that received a lactic acid treatment. Similar results were observed in the case of Salmonella Typhimurium (Fig. 2); however, counts appear to have de- FIGURE 1. Survival of E. coli O157:H7 in ground beef produced from outside rounds treated with water wash only (l), water wash plus prechill lactic acid spray (m), or water wash plus prechill plus postchill lactic acid sprays (m ). FIGURE 2. Survival of Salmonella Typhimurium in ground beef produced from outside rounds treated with water wash only (l), water wash plus prechill lactic acid spray (m), or water wash plus prechill plus postchill lactic acid sprays (m ).

62 CASTILLO ET AL. J. Food Prot., Vol. 64, No. 1 creased faster in ground beef made from rounds treated by water wash plus prechill lactic acid plus postchill lactic acid sprays. These results indicate that lactic acid may exhibit an extended antibacterial effect during storage, adding continued protection against the survival of pathogenic bacteria such as E. coli O157:H7 or Salmonella. A continued antimicrobial effect has been observed by others during storage of meat after spraying with lactic or acetic acid solutions. Kotula and Thelappurate (20) reported that agar plate counts and E. coli counts increased more rapidly on untreated steaks than on steaks treated with acetic or lactic acid. Similar results were reported by Dorsa et al. (14) for E. coli O157:H7, Listeria innocua, and Clostridium sporogenes on beef carcass surface tissue. In a study on the effects of lactic acid on calf carcasses and primal veal cuts (24), the agar plate counts of cold- or hot-boned primal cuts obtained from carcasses treated with lactic acid increased just 0.4 log 10 /cm 2 during a 14-day storage at 38C, whereas the agar plate counts of untreated controls increased by 2 log 10 /cm 2. In a study on the shelf life of strip loins sprayed with a hot (558C) 50:50 mixture of 2% lactic and 2% acetic acids, counts of psychrotrophic, aerobic, anaerobic, and lactic acid bacteria were signi cantly lower than those of nonsprayed loins during 84 days of storage at 218C (15). In conclusion, a signi cant bacterial reduction can be obtained on chilled carcass surfaces using a 4% solution of L-lactic acid at 558C sprayed for 30 s to deliver a total volume of 500 ml per carcass side. 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