Validation of Hot Water and Lactic Acid Sprays for the Reduction of Enteric Pathogens on the Surface of Beef Carcasses

Transcription

1 Validation of Hot Water and Lactic Acid Sprays for the Reduction of Enteric Pathogens on the Surface of Beef Carcasses G.R. Acuff, E. Cabrera-Diaz, L.M. Lucia Department of Animal Science, Texas A&M University, College Station, TX , USA PURPOSE To validate microbial interventions hot water and lactic acid spray for the reduction or elimination of enteric pathogens. INDICATOR ORGANISM A nonpathogenic Escherichia coli Biotype 1 strain was transformed to produce a green fluorescing protein (GFP), which also expressed ampicillin resistance properties (100 µg/l), at the Food Microbiology Laboratory at Texas A&M University (College Station, TX). This marker organism was designed to represent possible contamination of a carcass with enteric pathogens of fecal origin such as Salmonella or E. coli O157:H7. This indicator organism demonstrated identical thermal and acid resistance to E. coli O157:H7 INOCULUM PREPARATION Each slaughter day, feces were randomly collected from cattle in holding. Feces were dispensed in 10-g portion to individual stomacher bags. Ten mls of 12-h GFP E. coli cultures were transferred to each bag of feces (containing a concentration of ca. 7 log CFU/g). An identical set of bags containing feces not inoculated with the indicator organism was prepared with 10 mls of sterile 0.1% peptone water and mixed in the same manner as the inoculated bags. The uninoculated feces were used to evaluate the effect of treatments on traditional indicator organisms such as coliforms. SAMPLE COLLECTION On each of two slaughter days, eight cattle were harvested and dressed following USDA-FSIS-regulated commercial procedures. After trimming and prior to the hot water wash, the neck regions of each side were inoculated with either a fecal slurry or a fecal slurry containing the GFP E. coli. The surface was washed with potable water using a hand-held sprayer until visible feces was removed (~ 30 sec). Excise samples (10 cm 2 x 2 mm) were collected from the inoculated region of each carcass side before hot water treatment, after hot water treatment, and after lactic acid spray. All samples were packed in an insulated cooler with refrigerant packs and shipped to the Food Microbiology Lab at Texas A&M University. Additional data were also collected on the carcass surface, such as temperature before and after hot water, temperature before and after lactic acid spray

2 and ph before and after lactic acid spray. Other physical parameters were also collected, the concentration and temperature of the lactic acid spray, the time (sec) of the lactic acid spray, and time (min) of the hot water spray. MICROBIOLOGICAL ANALYSIS Coliform counts as well as counts of the indicator organisms were evaluated to establish the best means for validating the process. Petrifilm E. coli Count Plates were utilized for examination of samples inoculated with feces only. Counts of GFP E. coli from samples inoculated with feces containing GFP E. coli indicator organisms were determined by plating appropriate serial dilutions on tryptic soy agar (TSA) supplemented with ampicillin (100 µg/l). RESULTS AND DISCUSSION The hot water carcass wash was found to significantly reduce the coliform count as well as the indicator GFP E. coli. The minimum temperature observed was 124 o F and the maximum was found to be 165 o F with an average of 151 o F. The total time in spraying the hot water was very consistent over the two days of sampling and ranged from 16.0 to 18.3 min with an average of 17.3 min (Table 1). The concentration of the lactic acid ranged from 1.9 to 2.3 with an average of 2.1%. The lactic acid temperature was held constant at an average of 122 o F ( o F) and was sprayed from 32 sec to 126 sec with an average contact time of 69 sec (Table 2). The average carcass temperatures before hot water, after hot water, after lactic acid were 80 o F, 96 o F, and 84 o F respectively. The average ph of the carcass before and after hot water was 7.2 and 7.5 respectively while the ph dropped to an average of 3.5 after the addition of lactic acid (Table 3). The lactic acid carcass spray provided no significant reductions beyond that attained by hot water spray. In most cases on the non-inoculated feces, the hot water wash reduced counts to below the minimum detection level (0.7 log 10 /cm 2 ), preventing a subsequent validation of the lactic acid spray (Fig. 1). Reduction of GFP E. coli indicator organism by hot water treatment was consistent on all carcass sides. Hot water carcass sprays dramatically reduced the counts for coliforms on the inoculated carcass with mean log 10 /cm 2 reduction of 4.0. GFP E. coli followed similar trends with a 4.0 reduction (Table 4). The hot water carcass spraying procedure in this facility is apparently effective for reducing coliforms and the indicator GFP E. coli with a mean log reduction of 4.0 on a consistent basis. Although it was difficult to evaluate the lactic acid spray due to the effectiveness of the hot water treatment, both treatments together did reduce all indicator bacteria to below detectable limits. Previous research in this laboratory (see attached reprints) found coliforms to be slightly more resistant than enteric pathogens and reductions of the GFP indicator to be identical to that expected by enteric pathogens such as E. coli O157:H7 and Salmonella. Therefore, it is our conclusion that the intervention procedure used by this facility of a hot water spray at >150 o F for 9 min per side can obtain a 4-log reduction of enteric pathogens. The subsequent treatment of carcass sides with a lactic acid spray may provide additional assurance of reduction of pathogens; however, our data is unable to validate this assumption.

3 RECOMMENDATIONS It is possible that any hide-penetrating knife cuts made during dehiding could inoculate the carcass surface with enteric pathogens. Try to reduce contamination initially on carcass surface by tightening hygienic practices during dehiding, ie., sanitize knives frequently, especially after making initial cuts through the hide. Also, attempts should be made to be consistent in the application of hot water and lactic acid through standardizing of time and temperature parameters.

4 Table 1. Temperature and time of hot water wash application to beef carcass surfaces Temperature (ºF) Time (min) Min Max Average a a n = number of samples = 8

5 Table 2. Temperature, time, concentration and ph of lactic acid spray applied to beef carcass surfaces Temperature (ºF) a Time (sec) b Concentration (%) c phd Min Max Average a,c,d n = 2 (temperature, concentration and ph of lactic acid solution were measured once a day) b n = 23

6 Table 3. Temperature and ph of beef carcass surfaces before hot water wash, after hot water wash and after lactic acid spray application Temperature (ºF) ph Min Max Average a Min Max Average Before Hot Water After Hot Water After Lactic Acid a n = 84 (28 carcass sides, 3 measurements/side)

7 Table 4. Green fluorescent protein expressing (GFP) Escherichia coli and Coliforms counts on beef carcass surfaces before hot water wash, after hot water wash and after lactic acid spray application GFP-Escherichia coli Coliforms Min Max Avg a SD b Min Max Avg SD Before Hot Water After Hot Water After Lactic Acid a Average (n = 14) b Standard deviation

8 Table 5. Total count and reduction of GFP-Escherichia coli and Coliforms on beef carcass surfaces before hot water wash, after hot water wash and after lactic acid spray application Mean Log 10 Mean Log 10 Reduction BHW AHW ALA AHW ALA Combined GFP-Escherichia coli Coliforms BHW = before hot water wash AHW = after hot water wash ALA = after lactic acid spr

9 6 Fig. 1 Mean log 10 /cm 2 Escherichia coli Biotype 1 GFP and Coliform Counts on Beef Carcasses. 5 4 GFP Coliforms log CFU/cm Before Hot Water After Hot Water After Lactic Acid Treatment