Effect of Cold Temperature on Germicidal Efficacy of Quaternary Ammonium Compound, lodophor, and Chlorine on Listeria

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1 Journal of Food Protection, Vol. 56, No. 12, Pages (December 1993) Copyright, International Association of Milk, Food and Environmental Sanitarians 1029 Effect of Cold Temperature on Germicidal Efficacy of Quaternary Ammonium Compound, lodophor, and Chlorine on Listeria ERDAL U. TUNCAN ConAgra Frozen Foods, Columbia Analytical Laboratory, 409 Vandiver Drive, Bldg. 7, Suite 102, Columbia, Missouri (Received for publication April 12, 1993) ABSTRACT The germicidal efficacy of three common sanitizers (quaternary ammonium compound, iodophor, and chlorine) on Listeria (L. monocytogenes V7, L. monocytogenes Scott A,, and ) was studied using the suspension test method at various exposure temperature (2- C) and times ( min). The quaternary ammonium compound, iodophor, and chlorine solutions were prepared with sterile deionized water at concentrations of - ppm, ppm, and - ppm, respectively. All three sanitizers were effective (>5.0-log reduction in CFU/ml in 30 s) against Listeria at C regardless of their concentrations. The quaternary ammonium compound at - ppm and chlorine at - ppm inactivated a comparable number of Listeria cells at as they inactivated at C (i.e., cold temperature did not have any visible effect on the efficacy of the sanitizers). However, the efficacy of the quaternary ammonium compound and of the iodophor at ppm and lower concentrations decreased considerable as the exposure temperature decreased, yet the adverse effect of the cold temperature on the efficacy of the sanitizers was reversed via At cold temperatures, L. monocytogenes V7 and were inactivated by the quaternary ammonium compound to a lesser degree than L. monocytogenes Scott A and. Listeria monocytogenes, a pathogenic species of Listeria, has become a foodborne pathogen of great concern during the last decade due to the numerous outbreaks of listeriosis associated with food and resulting in high mortality (9). Since L. monocytogenes has become a foodborne pathogen of such concern, public and regulatory agencies have put great pressure on the food industry to produce Listeria-frtt products. However, Listeria is a very difficult bacterium to control in food processing plants because it is ubiquitous; hence, it is expected to be found in any type of food operation environment, it is able to grow below 4 C, and it has high tolerance, especially at cold temperatures, to adverse environmental conditions (2,5,6,9,14). Since Listeria can grow below refrigeration temperature and appears to survive better against adverse environmental conditions at cold temperatures, it is very likely to be found in cold areas of the food processing plants. Thus, it is critical to determine the effect of cold temperature on the efficacy of the sanitizers on Listeria. Previous studies have already shown the exposure (contact) temperature and time are important factors regarding the efficacy s (4,7,8,11,18), but most of these studies have been conducted at room and above room temperatures, and it was concluded that the sanitizers were more effective at higher temperatures (7,8,18). Some investigators included below-room temperatures in their studies and reported varying results depending upon the type of the sanitizer, test organism, sanitizer concentration, and the experimental condition of the study (4,10,12,16,17). Thus, the main focus of these studies was not the effect of cold temperature on the sanitizer efficacy. In addition, their results were based on only one or two exposure temperatures, exposure times, sanitizer types, or concentrations. In contrast to these previous studies, the focus of this study was the effect of cold temperature on the listericidal efficacy of various sanitizers (quaternary ammonium compound, iodophor, and chlorine) at various exposure temperatures (2- C) and times ( min) and at various sanitizer concentrations (12.5- ppm). The objective of the study was to determine the relationship between temperature and other variables such as exposure time and sanitizer concentration with regard to their effect on the efficacy of the sanitizers against Listeria. MATERIALS A METHODS Test cultures The test organisms used in the study were Listeria monocytogenes strain Scott A, Listeria monocytogenes strain V7, Listeria ivanovii, and Listeria innocua. The test organisms were grown in brain heart infusion broth (Difco, Detroit, MI) at 35 C by transferring the culture to a fresh brain heart infusion broth daily. The test cultures were prepared from the third generation of the test organisms by washing and suspending them in sterile saline solution. The test culture was either the individual test organisms or a pool of equal volumes of the cultures of the four test organisms in sterile saline solution. Sanitizing solutions The sanitizers tested in the study were quaternary ammonium compound (quat), containing 7.5% didecyl dimethyl ammonium chloride as the active ingredient, iodophor, containing 1.75% titratable iodine, and 12.5% sodium hypochlorite solution as the JOURNAL OF FOOD PROTECTION. VOL. 56, DECEMBER 1993

1030 TUNCAN chlorine (Chemland, Inc., Turlock, CA). The sanitizing solutions at designated concentrations were prepared in sterile deionized water according to the manufacturer's directions.

2 1030 TUNCAN chlorine (Chemland, Inc., Turlock, CA). The sanitizing solutions at designated concentrations were prepared in sterile deionized water according to the manufacturer's directions. The concentrations of the quat, iodophor, and chlorine solutions were tested with Direct Reading Titrator, Iodine Drop Count, and Chlorine Drop Count test kits, respectively (LaMotte Chemical Products Company, Chestertown, MA). Neutralizing solution A neutralizing solution was prepared from the following ingredients to neutralize the quat, iodophor, and chlorine at the end of the designated exposure time during the inactivation assay: 0.2% azolectin (lecithin, type II-s commercial grade, Sigma, St. Louis, MO), 1% vol/vol Tween 80 (Difco), 0.1% Na 2 S0 3.5H 2 0 (Matheson Coleman and Bell Manufacturing Chemists, Norwood, OH), 0.1% Bacto Peptone (Difco), 0.85% NaCl (Fisher Scientific, St. Louis, MO), and 1% 0. M potassium phosphate buffer (ph 7.2). The solution was prepared by mixing the first three ingredients at low heat and adding the rest of the ingredients after dissolving them in deionized water. The solution was then adjusted to the ph 7.2 with 0.1 N NaOH, brought to the predetermined volume with deionized water, dispensed into test tubes, and autoclaved at 121 C for 20 min. To determine the toxicity of the neutralizing solution to Listeria, the culture was suspended in the neutralizing solution and incubated at C for 30 min. The appropriate dilutions of the culture were prepared with 0.1% peptone dilution blanks before and after a 30-min incubation and plated in duplicate tryptose agar (TA; Difco) plates. The plates were counted following incubation at 35 C for 24 h. In addition, the cell concentrations of the control samples (the culture suspended in 0.1% peptone solution instead of in the neutralizing solution) were determined. The experiment was carried out with five replicate samples. To determine the efficacy of the neutralizing solution, the sanitizing solution was mixed with the neutralizing solution and challenged with the Listeria culture by incubating it at C for 15 min. The appropriate dilutions of the culture were prepared with 0.1% peptone dilution blanks before and after incubation and plated in duplicate TA plates. The plates were counted following incubation at 35 C for 48 h. In addition, the cell concentrations of the control samples (the culture suspended in neutralizing solution without sanitizing solution) were determined. Each experiment was carried out with five replicate samples. Inactivation assay The test culture was challenged with the sanitizing solution at designated concentrations and temperatures by pipetting 0.1 ml of the culture into the 9.9 ml sanitizing solution. At the end of the designated exposure time, the assay culture was diluted (1:10), and at the same time the reaction was stopped by mixing 1.0 ml of the assay culture with 9.0 ml neutralizing solution. After stopping the reaction, the serial dilutions of the assay culture were prepared with 0.1% peptone dilution blanks, and the appropriate dilutions were plated in duplicate TA plates using the pour plate technique. The plates were counted following incubation at 35 C for 48 h to determine the number of survivors. Experiments were carried out with duplicate samples and were repeated at least twice. In addition, duplicate control samples were tested for each experiment to determine the initial cell concentration of the assay culture. RESULTS Toxicity and efficacy of the neutralizing solution The toxicity of the neutralizing solution to Listeria (7.7 log CFU/ml) was tested at room temperature for 30-min exposure time. There was no observed decline in the viable cell numbers of the culture from the exposure of the culture to the neutralizing solution, which indicated that the neutralizing solution was not toxic to the Listeria culture (data not shown). The efficacy of the neutralizer on 300 ppm quat and chlorine and on ppm iodophor was also tested. Since the viable cell concentration of the culture (7.7 log CFU/ml) did not decrease from exposure to the sanitizers (data not shown), it was concluded that all three sanitizers were neutralized effectively by the neutralizing solution. Effect of cold temperature on the efficacy of quat The efficacy of the quat at - ppm was examined against Listeria ( log CFU/ml) at temperatures ranging from 2 to C. The test culture was either the individual test organisms or the pool of the test organisms (L. monocytogenes V7, L. monocytogenes Scott A,, and ). At 30-s exposure (contact) time, ppm quat inactivated more than 7.0 log CFU/ml of Listeria, regardless of the temperature (Table 1). The quat at ppm also inactivated more than 7.0 log units of Listeria, except that it was able to inactivate slightly less Listeria (6.5 log units) at (Table 1). The results were similar when the test organisms were tested individually (Table 2). These results suggest that the efficacy of the quat at ppm and above did not vary with temperature. TABLE 1. Effect of cold temperature on the efficacy of quaternary ammonium compound against Listeria." C Log 10 reduction in CFU/ml b a The pool of L. monocytogenes V7,, L. monocytogenes Scott A, and. However, the listericidal efficacy of the quat at and ppm decreased as the exposure temperature decreased (Table 1). Similar results were observed when the test organisms were tested individually (Table 2). The results presented in Tables 1 and 2 show that the decrease in Listeria inactivation due to cold temperature was stronger at lower concentrations. Another observation was that the efficacy of the quat was not the same against all test organisms (Table 2). At cold temperatures, the inactivation of L. monocytogenes V7 and was less than the inactivation of L. monocytogenes Scott A and. To determine whether the effect of cold temperature at 30-s exposure time could be reversed by increasing the exposure time, the efficacy of the ppm quat was examined at various exposure times between 0.5 and 15 min. As the exposure time increased, more Listeria was inactivated JOURNAL OF FOOD PROTFCTION, VOL. 56. DECEMBER 1993

3 EFFECT OF COLD TEMPERATURE ON SANITIZER EFFICIENCY 1031 TABLE 2. Effect of cold temperature on the efficacy of quaternary ammonium compound against various Listeria spp. TABLE 4. Effect of cold temperature on the efficacy of iodophor against Listeria." lest organism Log reduction in CFU/ml a C Log reduction in CFU/ml b C a Lm b Scott A At 30-s exposure time. b Listeria monocytogenes by the quat (Table 3). This indicates that the effect of cold temperature on the efficacy of the quat was reversible via Effect of cold temperature on the efficacy of iodophor The listericidal efficacy of the iodophor at ppm was examined at various temperatures between 2 and C against the pool of four test organisms. The cell concentration was log CFU/ml. The results presented in Table 4 show that the iodophor at C inactivated more than 7.0 log CFU/ml of TABLE 3. Effect of exposure time on the listericidal efficacy of quaternary ammonium compound and iodophor at various temperatures " The pool of L. monocytogenes V7,, L. monocytogenes Scott A, and. Listeria in 30 s, regardless of the sanitizer concentration. However, the iodophor inactivated less Listeria as the exposure temperature decreased. This decrease in Listeria inactivation varied with the iodophor concentration (Table 4). The relationship between the exposure time and the effect of cold temperature on the iodophor efficacy was also examined. According to the results presented in Table 3, the iodophor at 12.5 ppm inactivated much less Listeria at in 30 s than at C in 30 s, but it was able to inactivate as much Listeria at in 5 min as it did at C in 30 s. The adverse effect of cold temperature on the iodophor efficacy appeared to be completely reversible via Effect of cold temperature on the efficacy of chlorine The listericidal efficacy of the chlorine at - ppm was investigated against the pool of four test organisms between 2 and C. The cell concentration was log CFU/ml The chlorine at - ppm inactivated more than 7.0 log CFU/ml of Listeria at C in 30 s (Table 5). The results were the same at temperatures as low as. TABLE 5. Effect of cold temperature against Listeria." C on the efficacy of chlorine Log 10 reduction in CFU/ml b Time (min) 0.5 a b C Log reduction in CFU/ml C a The pool of L. monocytogenes V7, L innocua, L. monocyto genes Scott A, and Quaternary ammonium compound ( ppm). Iodophor (12.5 ppm). Not determined. DISCUSSION According to Chambers (3) and William (79), sanitizers are considered effective if they inactivate bacteria at least 5 log cycles in 30 s. Based on this criterion, the results of the present study indicate that the quat and chlorine at as low as ppm and the iodophor at as low as 12.5 ppm were effective against Listeria at room temperature. The effec- JOURNAL OF FOOD PROTECTION. VOL. 56. DECEMBER 1993

4 1032 TUNCAN tiveness of these three sanitizers against suspended Listeria cells was also noted by other investigators (13,15). Although the effect of cold temperature on the efficacy of the quat and iodophor was not visible at high concentrations (- ppm), the efficacy of both sanitizers declined noticeably due to cold temperature as their concentrations decreased. In contrast to the quat and iodophor, there was no observed decline on the listericidal efficacy of the chlorine (- ppm) as the contact temperature decreased from to (i.e., the chlorine efficacy appeared mot to be affected by cold temperature). These results concur with the results of previous studies in which it was reported that the effect of temperature could vary depending upon the type and concentration of the sanitizers. Overdahl and Zottala (17) reported that 10 ppm quat was able to inactivate less Staphylococcus haemolyticus and Bacillus spp. at 4 C than at C, but there was no visible difference in inactivation of S. haemolyticus by 10 ppm chlorine at 4 and C. According to Orth and Mrozek (16), a greater concentration of the quat was necessary at 5 C to inactivate a comparable number of Listeria cells inactivated at 20 C. Exposure time is also an important variable affecting the efficacy of the sanitizers (7,11). Dunsmore and Thomson (7) stated that a cold environment could limit the performance of detergents and sanitizers, but that the disadvantage of the cold temperature could be overcome by increasing the exposure time. For example, iodine (3-4 ppm) required 22 min at 3 C, but it required only 12 min at C to inactivate more that 5.0 log bacterial cells in drinking water (12). According to Collins (4), calcium hypochlorite at 3 ppm inactivated 4.0-log bacterial cells at 21 C in 4 min, but it required 10 min at 4.4C to inactivate 4.0-log bacterial cells. The results of the present study also suggest that the reduced efficacy of the quat and iodophor due to cold temperature could be reversed by increasing the exposure time. In fact, the adverse effect of cold temperature on the efficacy of the sanitizers was completely reversed by According to these results, cold temperature had a considerable effect on the efficacy of the quat and iodophor but not on the efficacy of the chlorine. The effect of cold temperature was probably overcome by greater germicidal potency of the chlorine and (or) by faster Listeria inactivation rate of the chlorine. As it has been previously reported (1), various species of bacteria may show varying resistance to the sanitizers. In the present study, less L. monocytogenes V7 and were inactivated at cold temperatures compared to L. monocytogenes Scott A and. This was likely due to the contribution of the greater quat resistance of L. monocytogenes V7 and. Thus, the effect of cold temperature on the efficacy of the quat appeared to be stronger against L. monocytogenes V7 and. These results suggest that the inherent resistance of the species and strain of Listeria toward a sanitizer should also be considered as a contributing factor to the effect of cold temperature on the efficacy of the sanitizer. The results of this study and of previous studies indicate that cold temperature can reduce the efficacy of the sanitizers substantially via decreasing the rate of inactivation of bacteria by the sanitizers. The effect of cold temperature on the sanitizer efficacy is dependent upon the type and concentration of the sanitizer, the resistance of the species, and strain of the bacteria to the sanitizer. Other factors such as presence of organic material, high or low ph, and the attachment of the bacterial cells to the surface have also been found to affect the efficacy s (8,10-12). In conclusion, the quat and iodophor can be ineffective against Listeria at cold temperatures even though they are effective against Listeria at room temperature. However, the effect of cold temperature can be avoided by increasing the exposure time or the concentration of the sanitizers. At first glance, it would appear that chlorine was the best sanitizer against Listeria at cold temperature. However, other factors can also affect efficacy, and they must be considered when selecting a sanitizer intended to eliminate cold-surviving organisms like Listeria. ACKNOWLEDGMENTS I would like to thank Dr. Sami Al-Hasani, Dr. Jim Canada, and Joel Zimmerman from ConAgra Frozen Foods Company for their input and support. I would also like to extend my appreciation to George Yonano and Milton Velibaba from Chemland, Inc., for their contributions to the study. REFERENCES 1. Best, M., M. E. Kennedy, and F. Coates Efficacy of a variety of disinfectants against Listeria spp. Appl. Environ. Microbiol. 54: Brackett, R. E Presence and persistence of Listeria monocytogenes in food and water. Food Technol. 42(4): Chambers, C. W A procedure for evaluating the efficiency of bactericidal agents. J. Milk Food Technol. 19: Collins, E. B Factors involved in the control of gelatinous curd defects of cottage cheese. II. Influence of ph and temperature upon the bactericidal efficiency of chlorine. J. Milk Food Technol. 18: Cox, L. J., J. Kleiss, J. L. Condier, C. Cordelena, P. Konkel, C. Pedrazzini, R. Beuner, and A. Siebenga Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: Doyle, M. P Effect of environmental factors and processing conditions on Listeria. Food Technol. 42(4): Dunsmore, D. G., and M. A. Thomson Bacteriological control of food equipment surfaces by cleaning systems. II. Sanitizer effects. J. Food Prot. 44: Dychdala, G. R Chlorine and chlorine compounds, pp In S. S. Block (ed.), Disinfection, sterilization, and preservation, 3rd ed. Lea and Febiger, Philadelphia, PA. 9. Farber, J. M., and P. I. Petcrkin Listeria monocytogenes, a foodbome pathogen. Microbiol. Rev. 55: Frank, J. F., and R. A. Koffi Surface-adherent growth of Listeria monocytogenes is associated with increased resistance to surfactant sanitizers and heat. J. Food Prot. 53: Giese, J. H Sanitation: the key to the food safety and public health. Food Technol. 45(12) Gottardi, W Iodine and iodine compounds, pp In S. S. Block (ed.), Disinfection, sterilization, and preservation, 3rd ed. Lea and Febiger, Philadelphia, PA. 13. Lopes, J. A Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeria monocytogenes. J. Dairy Sci. 69: Lovett, J., and R. M. Twedt Listeria. Food Technol. 42(4): McCarthy. S. A Attachment of Listeria monocytogenes to chitin and resistance to biocides. Food Technol. 46(12): JOURNAL OF FOOD PROTECTION, VOL. 56, DECEMBER 1993

5 EFFECT OF COLD TEMPERATURE ON SANITIZER EFFICIENCY Orth, R and H. Mrozek Is the control of Listeria, Campylobacter and Yersinia a disinfection problem? Fleischwirtsch. Int. 2: Overdahl, B. J., and E. A. Zottala Evaluation of selected sanitizers to control bacteria in a simulated sweet water coolant system. J. Food Prot. 54: Petrocii, A. N Surface-active agents: quaternary ammonium compounds, pp In S. S. Block (ed.), Disinfection, sterilization, and preservation, 3rd ed. Lea and Febiger, Philadelphia, PA. 19. William, S. (ed.) Disinfectants, p. 70. In Official methods of analysis of the association of official analytical chemists, 14th ed. Association of Official Analytical Chemists, Arlington, VA. JOURNAL OF FOOD PROTECTION, VOL. 56. DECEMBER 1993

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