Effect of Chemical Cleaning Agents and Commercial Sanitizers on ATP Bioluminescence Measurements

Transcription

1 86 Journal of Food Protection, Vol. 62, No. 1, 1999, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Research Note Effect of Chemical Cleaning Agents and Commercial Sanitizers on ATP Bioluminescence Measurements TRACY A. GREEN, SCOTT M. RUSSELL,* AND DANIEL L. FLETCHER Department of Poultry Science, The University of Georgia, Athens, Georgia , USA MS : Received 29 April 1998/Accepted 10 August 1998 ABSTRACT Nine chemical cleaning agents at three concentrations were studied to determine the effect on ATP bioluminescence measurements from pure ATP (PATP) and ATP from chicken exudate (CJATP). The nine commercial cleaning and sanitizing chemicals were concentrated foaming acid (FA), acid sanitizer (AS), iodine cleaner disinfectant (ZI), alkaline cleaner degreaser (PC), chlorinated alkaline cleaner (CA), chlorinated sanitizer (CS), quaternary ammonium (QA), antibiofilm agent (AB), and acidic peroxygen sanitizer (HP). Effect was reported as a percent change from the log 10 relative light unit (LRLU) measurements of the control groups. All cleaners and sanitizers were tested at one-tenth of the manufacturer s recommended level (MRL), MRL, and two times MRL. FA, PC, and CA at all three concentrations significantly decreased PATP and CJATP LRLU. AS decreased PATP and CJATP LRLU at 200 and 400 ppm quaternary ammonium. ZI decreased PATP LRLU at MRL or greater, while CJATP LRLU were decreased by all concentrations of ZI tested. CS decreased PATP LRLU in a dosedependant manner; however, for CJATP, LRLU decreased slightly at the two lower concentrations but were not affected by 1,200 ppm CS. QA at MRL or above for PATP or at all concentrations for CJATP significantly increased LRLU. AB decreased LRLU at all concentrations tested for PATP or at MRL or greater for CJATP. HPA at MRL or greater for PATP or at all concentrations for CJATP significantly reduced LRLU. These results demonstrate that commercial sanitizers and cleansers may squelch or increase LRLU measurements when the chemical comes into direct contact with the ATP bioluminescence reagents. Hence, when using ATP bioluminescence as a means of determining sanitary quality of food-processing equipment, it is essential to consider the type and concentration of chemical cleaner or sanitizer being used on the equipment prior to testing. The United States Department of Agriculture Food Safety and Inspection Service (USDA FSIS) recently published The Pathogen Reduction/Hazard Analysis and Critical Control Point System (HACCP) Final Rule (9). This ruling is designed to increase safety of meat and poultry by monitoring and controlling processing steps that affect biological, chemical, or physical hazards associated with production. Prior to the initiation of HACCP, the USDA FSIS required all red meat- and poultry-processing facilities to develop and implement a Sanitation Standard Operating Procedures (SSOPs) program that describes the facilities procedures for removing bacteria and organic material from food contact surfaces (4, 6, 9). SSOPs generally include a microbiological method to verify that the processing plant and equipment have been properly cleaned and sanitized. Because it is expensive and time consuming to identify and enumerate specific pathogenic and spoilage bacteria, most poultry-processing facilities evaluate equipment sanitation by enumerating indicator bacteria (4). Indicator bacteria are usually enumerated using aerobic plate counts (APC), RO- DAC plates, or Petrifilm (5) that require incubation times of 24 to 48 h and provide retrospective assessments of san- * Author for correspondence Tel: ; Fax: ; srussell@uga.cc.uga.edu. itation that cannot be used to evaluate plant cleanliness prior to initiation of production (3, 5, 10). There has been considerable interest in developing rapid microbiological enumeration methods to eliminate the time delay to acquire results. ATP bioluminescence has gained popularity for monitoring sanitation because processing equipment can be evaluated in minutes rather than days (1, 5, 6). ATP bioluminescence is based on detection and quantification of ATP using the luciferase enzyme and the luciferin cofactor (8). Hydrolysis of ATP by luciferase produces yellow-green light, which is detected using a luminometer and reported as relative light units (RLUs) or log 10 RLU (LRLU) (5, 8). Because one ATP molecule produces one photon of light, light intensity from the reaction is proportional to the amount of ATP in the sample (8). Equipment surfaces with high APC values also have high RLU values, presumably because they have a higher concentration of microorganisms and thus more ATP (3, 5). Processing residues (blood and tissue) on equipment may also contribute to increases in RLU values because luciferase reacts with both eukaryotic and prokaryotic ATP (1, 3, 5, 6). Processing residues are indicative of insufficient sanitation and can be detected using ATP bioluminescence, while APC is useful only for enumerating microorganisms left on the equipment (3, 5, 8). If processing equipment fails the bioluminescence test due to high RLU values, re-

2 J. Food Prot., Vol. 62, No. 1 EFFECT OF CLEANERS AND SANITIZERS ON ATP BIOLUMINESCENCE 87 gardless of the ATP source, it should be recleaned prior to the initiation of processing (1). ATP bioluminescence readings may be affected by exposing reaction components (i.e., firefly luciferase, luciferin cofactor, or ATP) to chemical compounds. Using pure ATP, Velazquez and Fiertag (11) reported that 0.01% alkaline foam cleanser increased ATP measurements and significantly decreased RLU measurements at concentrations of 0.1 to 10%. The authors also reported that RLU measurements decreased when 0.3 to 10% acid foam cleaner, 1 to 5% D-limolene, 0.1% and higher commercial (12.5%) sodium hypochlorite, and 1% and higher (5.25%) sodium hypochlorite (bleach) were used (11). RLU readings increased in the presence of 0.01% commercial and 0.01% household sodium hypochlorite and 25 to 800 ppm quaternary ammonium sanitizer (11). Because sanitizers have been shown to influence ATP bioluminescence readings, it is possible that commonly used cleansers may have similar influences on RLU output. The purpose of this study was to determine the effect of nine chemical cleaning agents and commercial sanitizers on ATP bioluminescence measurements using different ATP sources. MATERIALS AND METHODS Reaction mixture. Two sources of ATP, pure ATP (PATP) and ATP extracted from broiler carcass exudate (CJATP), were exposed to nine commercial chemical cleansers or commercial sanitizers at three concentrations in each of six replicate trials. Control groups were identical to treatment groups except deionized water was substituted for the chemicals. Ten samples each of PATP and CJATP were analyzed to obtain control LRLU, and three samples were analyzed for each concentration ATP source combination of the nine test chemicals in each of the six repetitions. To test each sample, 400 l of ATP-releasing reagent (releases ATP from cells) was added to a cuvette followed by the addition of 400 l of the cleaning or sanitizing chemical (or 400 l of deionized water for controls). ATP (100 l PATPor70 l CJATP) was then added to each cuvette, mixed thoroughly, and allowed to stand for 5 min prior to bioluminescence measurement. Time required for the ATP to be released was standardized at 5 min because a preliminary study showed that the length of time the carcass exudate is exposed to the ATP-releasing reagent influenced the RLU measurements. ATP sources. PATP was obtained from the ATP-positive control included in the Celsis Hygiene Monitoring Kit (Celsis, St. Louis, Mo.). Freeze-dried pure ATP along with the hygiene monitoring kit were stored at 4 C, and ATP was reconstituted immediately prior to the initiation of each repetition. Broiler carcass exudate was collected from a local poultryprocessing plant from plastic boxes in which broiler front halves were stored. Exudate was collected in sterile 250-ml nalgene bottles and stored on ice for transportation to the laboratory (approximately 1 h). Exudate was mixed thoroughly and filtered through Whatman qualitative 15-cm filter paper. One milliliter of the filtrate was transferred to 1-ml microcentrifuge tubes and frozen at 27 C. A preliminary study demonstrated that ATP values decreased significantly when the carcass exudate was allowed to remain at room temperature for 1 h or more. Thus, one tube of carcass TABLE 1. Type, abbreviation, and treatment concentrations of chemical sanitizers or cleansers Chemical Abbreviation Treatment concentration Foaming acid Acid sanitizer FA AS 0.63, 6.3, 12.6% 20, 200, 400 ppm Iodine cleaner disinfectant ZI 7.5, 75, 150 ppm Alkaline cleaner degreaser PC 0.63, 6.3, 12.6% Chlorinated, highly alkaline cleaner CA 0.63, 6.3, 12.6% Chlorinated sanitizer CS 60, 600, 1,200 ppm Quaternary ammonium QA 26.7, 267, 534 ppm Antibiofilm agent AB 0.1, 1.0, 2% Acidic peroxygen sanitizer a HP, hydrogen peroxide. b PAA, peroxyacetic acid. HPA 64.4, 644, 1,288 ppm NP a 15.2, 152, 304 ppm PAA b exudate was thawed to determine the control values for each ATP source in each of the six repetitions. One tube of exudate was also thawed for RLU measurements for each of the nine test chemicals per repetition. Test chemicals. Test chemical solutions were prepared by mixing commercial liquids in deionized water. The type, brand, abbreviation, and treatment concentrations of each chemical are presented in Table 1. Test concentrations were determined by using the manufacturer s recommended level as the middle concentration. The lowest concentrations used were one-tenth the recommended concentration, while the highest chemical concentrations used were two times the recommended levels for each chemical. RLU determination. RLU s were determined using the Celsis Optocomp 1 luminometer (Celsis). The Optocomp 1 automatically injects 200 l of luciferin luciferase reagent (Hygiene Monitoring Kits, Celsis) when a sample cuvette is placed into the instrument. After a 0.5-s delay, the luminometer measures the light produced during the ATP bioluminescence reaction. For this study, data are reported and analyzed as log 10 RLU (LRLU). Statistical analysis. The experimental design was factorial for replicate, ATP source, test chemicals, and concentration, using triplicate samples for a total of 972 samples. Treatment LRLU were analyzed and reported as percentages of control LRLU values ([log 10 RLU test chemical concentration/ log 10 RLU control] 100) for each test chemical concentration and ATP source. Effects of each sanitizer on LRLU were determined using the analysis of variance option and means were separated using Duncan s multiple range test option of the generalized linear models procedure of the SAS/STAT program (7). Main effects of test chemical, replicate, and test chemical by replicate interaction were determined using residual error within each level of each different test chemical. When the treatment by replicate interaction was significant (P 0.05), it was used as the test error to determine treatment differences. RESULTS AND DISCUSSION Results for the effect of each test chemical concentration on PATP and CJATP LRLU measurements are pre-

3 88 GREEN ET AL. J. Food Prot., Vol. 62, No. 1 FIGURE 1. Effect of chemicals on ATP bioluminescence measurements from pure ATP. sented in Figures 1 and 2, respectively, as a percentage of the control LRLU values (100%). Concentrated foaming acid (FA) at levels of 0.63, 6.3, and 12.6% significantly reduced (P 0.05) LRLU by 52, 71, and 71% for PATP and 40, 64, and 65% for CJATP. Velazquez and Fiertag (11) reported similar results using a different commercial FA cleaner in which treatment with 0.4% caused a 30% reduction of RLU measurements, while treatments with 1, 2, 3, 4, and 10% FA caused a 90% reduction in RLU measurements. Acid sanitizer (AS) LRLU for PATP or CJATP at 20 ppm did not differ from the control measurements; however, 200 and 400 ppm AS significantly (P 0.05) reduced LRLU by 5 and 33% for PATP, and 6 and 25% for CJATP, respectively. Iodine cleaner disinfectant (ZI) at 75 and 150 ppm (titratable iodine) significantly (P 0.05) reduced LRLU by 26 and 51% for PATP, and 19 and 41% for CJATP, while 7.5 ppm ZI had no effect (P 0.05) on LRLU measurements for PATP but decreased (P 0.05) LRLU for CJATP by 6%. Alkaline cleaner degreaser (PC) at levels of 0.63, 6.3, and 12.6% significantly (P 0.05) reduced LRLU for PATP and CJATP by 17, 69, and 72%, and 7, 61, and 70%, respectively. When the different ATP sources were treated with chlorinated, highly alkaline cleaner (CA) at levels of 0.63, 6.3, and 12.6%, a significant (P 0.05) reduction in LRLU measurements was observed for both PATP and CJATP (15, 60, and 68% and 9, 53, and 54%, respectively). Using a similar commercial alkaline foam cleaner at concentrations of 0.2 to 10%, Velazquez and Fiertag (11) reported similar reductions in PATP measurements. However, the authors reported a 15% increase in RLU measurements when reaction components were exposed to 0.01% of the alkaline foam cleaner (11). Results showed that use of a chlorinated sanitizer (CS 12.5%) at concentrations of 60, 600, and 1,200 ppm (available chlorine) significantly (P 0.05) reduced LRLU for PATP by 2, 7, and 12%. A significant (P 0.05) reduction of 5 and 4% was observed for CJATP at 60 and 600 ppm CS; however, 1,200 ppm CS did not change CJATP LRLU measurements when compared to CJATP controls. These findings agree with those reported by Velazquez and Fiertag (11) when they used similar concentrations of commercial sodium hypochlorite and measured PATP. RLUs were slightly enhanced when the reaction components were exposed to low levels (0.01%) of commercial sodium hypochlorite (11). Although the authors reported higher reductions in RLU by commercial sodium hypochlorite, the concentration-dependent squelching effects demonstrated by this sanitizer appears to be a common factor when PATP is used as the ATP source (11). Surprisingly, in the present study, the highest concentration (1,200 ppm active chlorine) of sanitizer used had no effect on LRLU measurements for CJATP. Treatment with a quaternary ammonium disinfectant (QA) significantly (P 0.05) increased LRLU for PATP by 3 and 7% at 267 and 534 ppm, respectively. QA at 26.7, 267, and 534 ppm significantly (P 0.05) increased LRLU readings for CJATP by 3, 5, and 10%, respectively. Similar enhancement effects by QA on ATP measurements were reported by Velazquez and Fiertag (11). Antibiofilm agent (AB) at 1 and 2% significantly re-

4 J. Food Prot., Vol. 62, No. 1 EFFECT OF CLEANERS AND SANITIZERS ON ATP BIOLUMINESCENCE 89 FIGURE 2. Effect of chemicals on ATP bioluminescence measurements from processing exudate ATP. duced LRLU by 53 and 53%, and 43 and 51% for PATP and CJATP, respectively. AB at 0.1% significantly (P 0.05) reduced PATP LRLU by 4% but did not significantly (P 0.05) effect CJATP LRLU readings. Acidic peroxygen sanitizer (HPA) concentrations of 644 ppm hydrogen peroxide (HP) 152 ppm peroxyacetic acid (PAA) and 1,288 ppm HP 304 ppm PAA significantly (P 0.05) reduced LRLU measurements by 6 and 31%, and 35 and 57% for PATP and CJATP, respectively. HPA at 64.4 ppm HP 15.2 ppm PAA did not significantly (P 0.05) affect PATP LRLU, while the same level decreased LRLU measurements for CJATP by 21%. Green et al. (2) reported that HP at 0.5% (5,000 ppm) and 1% (10,000 ppm) decreased PATP LRLU by 3 and 40%, respectively. In the present study, PATP LRLU reductions were similar to those reported by Green et al. (2) when HP concentrations were similar; however, when HPA containing similar concentrations of HP was exposed to samples containing CJATP, LRLU reductions were much greater (35 and 57%) than those reported by Green et al. Higher LRLU reductions in the present study may be due to the action of the PAA component of HPA in the presence of organic material (protein, fat, lipid membranes). Sanitizers and cleansers are formulated to kill microorganisms and/or remove processing residues from foodprocessing equipment or food contact surfaces. Many sanitizers and cleansers contain components that breakdown or destroy organic material such as fats, proteins, and biological membranes. This study demonstrates that exposing ATP bioluminescence reaction components directly to sanitizers and cleansers may significantly reduce LRLU measurements. Of the nine chemicals tested in the present study, QA was the only chemical that increased LRLU measurements. Similar results were obtained for the two ATP sources (PATP and CJATP) when exposed to the different chemicals; however, HPA (hydrogen peroxide and peroxyacetic acid sanitizer) caused larger LRLU reductions in ATP measurements from organic (CJATP) sources of ATP as opposed to the PATP source. ACKNOWLEDGMENTS This study was supported in part by state and Hatch funds allocated to the Georgia Agricultural Experiment Station. Appreciation is extended to Celsis, Zep Manufacturing, and Sterilix for their generous support, which included the Optocomp 1 Luminometer, Hygiene Monitoring Kits, technical assistance, test chemicals, and supplies. The authors thank Mark Carter for his generosity and technical assistance. REFERENCES 1. Cutter, C. N., W. J. Dorsa, and G. R. Siragusa A rapid microbial ATP bioluminescence assay for meat carcasses. Dairy Food Environ. Sanit. 16: Green, T. A., S. M. Russell, and D. L. Fletcher Effect of chemical sanitizing agents on ATP bioluminescence. J. Food Prot. 61(8): Kyriakides, A ATP bioluminescence applications for microbiological quality control in the dairy industry. J. Soc. Dairy Technol. 45: Marriott, N. J Principles of food sanitation. Van Nostrand Reinhold, New York.

5 90 GREEN ET AL. J. Food Prot., Vol. 62, No Russell, S. M ATP bioluminescence: it s portable and real time. Broiler Industry August: Russell, S. M Sanitation procedures and HACCP. Broiler Industry October: SAS Institute, SAS/STAT guide for personal computers, version 7 edition. SAS Institute, Inc., Cary, N.C. 8. Stanley, P. E A concise beginner s guide to rapid microbiology using adenosine triphosphate and luminescence, p In P. E. Stanley, B. J. McCarthy, and R. Smither (ed.), ATP luminescence. Blackwell Scientific Publications, Cambridge, Mass. 9. USDA FSIS Pathogen reduction; hazard analysis and critical control point (HACCP) systems. Fed. Reg. 61(144): Vanderzant, C., and D. F. Splittstoesser (ed.) Colony count methods, p In Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health Association, Washington, D.C. 11. Velazquez, M., and J. Fiertag Quenching and enhancement effects of ATP extractants, cleansers, and sanitizers on the detection of the ATP bioluminescence signal. J. Food Port. 60: