B. CEREUS BİYOFİLMLERİNİN SİTRİK ASİT UYGULAMALARI İLE KONTROLÜ
Yıl 2018,
Cilt: 43 Sayı: 4, 605 - 616, 15.06.2018
Meltem Yesilcimen Akbas
,
Taner Şar
Öz
Bu çalışmada, önemli bir gıda patojeni olan B. cereus
vejetatif hücrelerinin ve sporlarının mikrotitrasyon plaklarında, glukoz ve süt
içeren besiyerleri kullanılarak (TSBG ve TSBS)
oluşturduğu 24-72 saatlik biyofilmlerinin, %2 sitrik asit ve 200 ppm klor
uygulamaları ile önlenmesi ve ortadan kaldırılması araştırılmıştır. B.
cereus vejetatif hücrelerinin TSBG ve TSBS
besiyerleri kullanıldığında oluşan biyofilmlerinin, sitrik asit uygulamaları
ile %59 oranında önlendiği ve %38-63 oranlarında giderildiği belirlenmiştir. B.
cereus spor biyofilmlerinin ise, %56 oranında önlenebildiği ve %40-56
oranlarında giderilebildiği görülmüştür. Sitrik asit uygulamasının (%2) oluşan B.
cereus vejetatif hücrelerinin ve sporlarının biyofilmleri üzerinde klor
kadar etkili olabildiği tespit edilmiştir.
Kaynakça
- Abee, T., Kovács, Á.T., Kuipers, O.P., Van der Veen, S. (2011). Biofilm formation and dispersal in Gram-positive bacteria. Curr Opin Biotechnol, 22(2): 172-179. Abraha, A., Bikila, T., Alemu, S., Muktar, Y. (2017). Bacillus cereus isolation and load from raw cow milk sold in Markets of Haramaya District, eastern Ethiopia. Int. J. Food Contam, 4(1): 15.
- Akbas, M.Y., Cag, S. (2016). Use of organic acids for prevention and removal of Bacillus subtilis biofilms on food contact surfaces. RVCTA, 22(7): 587-597.
- Akbas, M.Y., Kokumer, T. (2015). The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments. Int J Food Sci Technol, 50(7): 1666-1672.
- Almasoud, A., Hettiarachchy, N., Rayaprolu, S., Babu, D., Kwon, Y.M., Mauromoustakos, A. (2016). Inhibitory effects of lactic and malic organic acids on autoinducer type 2 (AI-2) quorum sensing of Escherichia coli O157: H7 and Salmonella typhimurium. LWT-Food Sci Technol, 66: 560-564.
- Almasoud, A., Hettiarachchy, N., Rayaprolu, S., Horax, R., Eswaranandam, S. (2015). Electrostatic spraying of organic acids on biofilms formed by E. coli O157: H7 and Salmonella Typhimurium on fresh produce. Food Res Int., 78: 27-33.
- Austin, J.W., Berferin, G. (1995). Development of bacterial biofilms in dairy processing lines. J Dairy Res., 62: 509-519.Bennet, R.W., Belay, N. (2001). Bacillus cereus. In Compendium of methods for the microbiological examination of food, Downes, F.P., Ito, K. (Eds.), 4th edition, American Public Health Association, Washington, DC, Chapter 32, pp. 311–316.
- Bremer, P.J., Fillery, S., McQuillan, A.J. (2006). Laboratory scale clean-in-place (CIP) studies on the effectiveness of different caustic and acid wash steps on the removal of dairy biofilms. Int J Food Microbiol, 106(3): 254-262.
- Burgess, S.A., Lindsay, D., Flint, S.H. (2010). Thermophilic bacilli and their importance in dairy processing. Int J Food Microbiol, 144(2): 215-225.
- Carlin, F., Brillard, J., Broussolle, V., Clavel, T., Duport, C., Jobin, M., Guinebretiere, M.H., Auger, S., Sorokine, A., Nguyen-The, C. (2010). Adaptation of Bacillus cereus, an ubiquitous worldwide-distributed foodborne pathogen, to a changing environment. Food Res. Int., 43(7): 1885-1894.
- Corcoran, M., Morris, D., De Lappe, N., O'connor, J., Lalor, P., Dockery, P., Cormican, M. (2014). Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials. Appl Environ Microbiol, 80: 1507-1514.
- Faille, C., Lequette, Y., Ronse, A., Slomianny, C., Garénaux, E., Guerardel, Y. (2010). Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: consequences on their ability to adhere to stainless steel. Int J Food Microbiol, 143(3): 125-135.
- Faot, F., Cavalcanti, Y.W., e Bertolini, M.D.M., de Rezende Pinto, L., da Silva, W.J., Cury, A.A.D.B. (2014). Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly (methyl methacrylate): effects on residual biofilm and recolonization process. BMC Oral Health, 14(1): 77.
- Flemming, H.C., Wingender, J., Mayer, C., Korstgens, V., Borchard, W. (2000). Cohesiveness in biofilm matrix polymers. In Symposia-Society for General Microbiology, Cambridge; Cambridge University Press, pp. 87-106.
- Flint, S.H., Bremer, P.J., Brooks, J.D. (1997). Biofilms in dairy manufacturing plant description, current concerns and methods of control. Biofouling, 11(1): 81-97.
- Ghandbari, E.H. (1983). Reactions of Chlorine and Chlorine Dioxide with Free Fatty Acids, Fatty Acid Esters, and Triglycerides. In Water Chlorination: Environmental Impact and Health Effects, Vol. 4 ((Jolley, R.L., Brungs, W.A., Cotruvo, J.A., Cumming, R.B., Mattice, J.S., Jacobs, V.A., Eds.). Ann Arbor Sci. Publ., Ann Arbor. Mich, 167-180.
- Gómez-López, V.M., Marín, A., Medina-Martínez, M.S., Gil, M.I., Allende, A. (2013). Generation of trihalomethanes with chlorine-based sanitizers and impact on microbial, nutritional and sensory quality of baby spinach. Postharvest Biol Technol., 85: 210-217.
- Granum, P.E., Lund, T. (1997). Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett, 157(2), 223-228.
- Grutsch, A.A., Nimmer, P.S., Pittsley, R.H., McKillip, J.L. (2018). Bacillus spp. as Pathogens in the Dairy Industry. Foodborne Diseases, 193-211.
- Koutina, G., Skibsted, L.H. (2015). Calcium and phosphorus equilibria during acidification of skim milk at elevated temperature. Int Dairy J., 45: 1-7.
- Kreske, A.C., Ryu, J.H., Pettigrew, C.A., Beuchat, L.R. (2006). Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm. J Food Prot, 69(11): 2621-2634.
- Lindsay, D., Brözel, V.S., Mostert, J.F., Von Holy, A. (2000). Physiology of dairy-associated Bacillus spp. over a wide pH range. Int J Food Microbiol., 54(1): 49-62.
- Lücking, G., Stoeckel, M., Atamer, Z., Hinrichs, J., Ehling-Schulz, M. (2013). Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage. Int J Food Microbiol., 166(2): 270-279.
- Marchand, S., De Block, J., De Jonghe, V., Coorevits, A., Heyndrickx, M., Herman, L. (2012). Biofilm formation in milk production and processing environments; influence on milk quality and safety. Comp Rev Food Sci Food Safety, 11(2): 133-147.
- Mittelman, M. W. (1998). Structure and functional characteristics of bacterial biofilms in fluid processing operations. J Dairy Sci., 8(10), 2760-2764.
- Neal-McKinney, J.M., Lu, X., Duong, T., Larson, C.L., Call, D.R., Shah, D.H., Konkel, M.E. (2012). Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLoS One, 7(9): e43928.
- Noss, C.I., Dennis, W.H., Olivieri, V.P., 1983. Reactivity of Chlorine Dioxide with Nucleic-acids and Proteins. In Water Chlorination: Environmental Impact and Health Effects, Vol. 4 ((Jolley, R.L., Brungs, W.A., Cotruvo, J.A., Cumming, R.B., Mattice, J.S., Jacobs, V.A., Eds.). Ann Arbor Sci. Publ., Ann Arbor. Mich, 1077–1086.
- Ntrouka, V., Hoogenkamp, M., Zaura, E., van der Weijden, F. (2011). The effect of chemotherapeutic agents on titanium‐adherent biofilms. Clin Oral Implants, 22(11): 1227-1234.
- Organji, R.S., Abulreesh, H.H., Elbanna, K., Osman, G.E.H., Khider, M. (2015). Occurrence and characterization of toxigenic Bacillus cereus in food and infant feces. Asian Pac J Trop Biomed., 5(7): 515-520.
- Oulahal, N., Brice, W., Martial, A. Degraeve, P. (2008). Quantitative analysis of survival of Staphylococcus aureus or Listeria innocua on two types of surfaces: polypropylene and stainless steel in contact with different dairy products. Food Control, 19: 178-185.
- Pagedar, A., Singh, J. (2012). Influence of physiological cell stages on biofilm formation by Bacillus cereus of dairy origin. Int Dairy J., 23(1): 30-35.
- Parkar, S.G., Flint, S.H., Palmer, J.S., Brooks, J.D. (2001). Factors influencing attachment of thermophilic bacilli to stainless steel. J Appl Microbiol, 90(6): 901-908.
- Ramos-Villarroel, A.Y., Martín-Belloso, O., Soliva-Fortuny, R. (2015). Combined effects of malic acid dip and pulsed light treatments on the inactivation of Listeria innocua and Escherichia coli on fresh-cut produce. Food Control, 52: 112-118.
- Ricke S.C. (2003). Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sciences, 82: 632-639.
- Ryu, J.H., Beuchat, L.R. (2005). Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid–based sanitizer. J Food Prot, 68(12), 2614-2622.
- Sadiq, R., Rodriguez, M.J. (2004). Disinfection by-products (DBPs) in drinking water and predictive models for their occurrence: A review. Sci Total Environ. 321: 21-46.
- Sánchez, G., Elizaquível, P., Aznar, R., Selma, M.V. (2015). Virucidal effect of high power ultrasound combined with a chemical sanitizer containing peroxyacetic acid for water reconditioning in the fresh-cut industry. Food Control, 52: 126-131.
- Schaeffer, A.B., Fulton, M.D. (1933). A simplified method of staining endospores. Science, 77(1990): 194-194.
- Simões, M., Simoes, L.C., Vieira, M.J. (2010). A review of current and emergent biofilm control strategies. LWT-Food Sci Technol, 43(4): 573-583.
- Stepanović, S., Vuković, D., Dakić, I., Savić, B., Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods, 40(2): 175-179.
- Te Giffel, M.C., Beumer, R.R., Leijendekkers, S., Rombouts, F.M. (1996). Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiology, 13(1), 53-58.
- Van Haute, S., Sampers, I., Holvoet, K., Uyttendaele, M. (2013). Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing. Appl Environ Microbiol, 79(9): 2850-2861.
- Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J. (2005). Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl Environ Microbiol, 71(9): 5022-5028.
- Waters, B.W., Hung, Y.C. (2014). The effect of organic loads on stability of various chlorine‐based sanitisers. Int J Food Sci Technol, 49(3): 867-875.
- Zhang, Q.Q., Ye, K.P., Juneja, V.K., Xu, X. (2017). Response surface model for the reduction of Salmonella biofilm on stainless steel with lactic acid, ethanol, and chlorine as controlling factors. Journal of Food Safety, 37(3).1-7.
CONTROL OF B. CEREUS BIOFILMS BY CITRIC ACID TREATMENTS
Yıl 2018,
Cilt: 43 Sayı: 4, 605 - 616, 15.06.2018
Meltem Yesilcimen Akbas
,
Taner Şar
Öz
In this work, the prevention and removal of biofilm
formations of B. cereus vegetative cells and spore formations by citric
acid (2%) and chlorine (200 ppm) treatments on microtitration plates were
investigated. The biofilms were produced in the presence of glucose and milk
(TSBG and TSBM) for 24-72 hours. B. cereus
biofilms, formed by vegetative cells in TSBG and TSBM
were inhibited by citric acid treatments by up to 59% and removed about 38-63%,
respectively. However, biofilms of B. cereus spores were prevented about
56% and removed by 40-56%. It was shown that citric acid treatment (2%) could
be as effective as chlorine treatment for biofilms of B. cereus
vegetative cells and spores.
Kaynakça
- Abee, T., Kovács, Á.T., Kuipers, O.P., Van der Veen, S. (2011). Biofilm formation and dispersal in Gram-positive bacteria. Curr Opin Biotechnol, 22(2): 172-179. Abraha, A., Bikila, T., Alemu, S., Muktar, Y. (2017). Bacillus cereus isolation and load from raw cow milk sold in Markets of Haramaya District, eastern Ethiopia. Int. J. Food Contam, 4(1): 15.
- Akbas, M.Y., Cag, S. (2016). Use of organic acids for prevention and removal of Bacillus subtilis biofilms on food contact surfaces. RVCTA, 22(7): 587-597.
- Akbas, M.Y., Kokumer, T. (2015). The prevention and removal of biofilm formation of Staphylococcus aureus strains isolated from raw milk samples by citric acid treatments. Int J Food Sci Technol, 50(7): 1666-1672.
- Almasoud, A., Hettiarachchy, N., Rayaprolu, S., Babu, D., Kwon, Y.M., Mauromoustakos, A. (2016). Inhibitory effects of lactic and malic organic acids on autoinducer type 2 (AI-2) quorum sensing of Escherichia coli O157: H7 and Salmonella typhimurium. LWT-Food Sci Technol, 66: 560-564.
- Almasoud, A., Hettiarachchy, N., Rayaprolu, S., Horax, R., Eswaranandam, S. (2015). Electrostatic spraying of organic acids on biofilms formed by E. coli O157: H7 and Salmonella Typhimurium on fresh produce. Food Res Int., 78: 27-33.
- Austin, J.W., Berferin, G. (1995). Development of bacterial biofilms in dairy processing lines. J Dairy Res., 62: 509-519.Bennet, R.W., Belay, N. (2001). Bacillus cereus. In Compendium of methods for the microbiological examination of food, Downes, F.P., Ito, K. (Eds.), 4th edition, American Public Health Association, Washington, DC, Chapter 32, pp. 311–316.
- Bremer, P.J., Fillery, S., McQuillan, A.J. (2006). Laboratory scale clean-in-place (CIP) studies on the effectiveness of different caustic and acid wash steps on the removal of dairy biofilms. Int J Food Microbiol, 106(3): 254-262.
- Burgess, S.A., Lindsay, D., Flint, S.H. (2010). Thermophilic bacilli and their importance in dairy processing. Int J Food Microbiol, 144(2): 215-225.
- Carlin, F., Brillard, J., Broussolle, V., Clavel, T., Duport, C., Jobin, M., Guinebretiere, M.H., Auger, S., Sorokine, A., Nguyen-The, C. (2010). Adaptation of Bacillus cereus, an ubiquitous worldwide-distributed foodborne pathogen, to a changing environment. Food Res. Int., 43(7): 1885-1894.
- Corcoran, M., Morris, D., De Lappe, N., O'connor, J., Lalor, P., Dockery, P., Cormican, M. (2014). Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials. Appl Environ Microbiol, 80: 1507-1514.
- Faille, C., Lequette, Y., Ronse, A., Slomianny, C., Garénaux, E., Guerardel, Y. (2010). Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: consequences on their ability to adhere to stainless steel. Int J Food Microbiol, 143(3): 125-135.
- Faot, F., Cavalcanti, Y.W., e Bertolini, M.D.M., de Rezende Pinto, L., da Silva, W.J., Cury, A.A.D.B. (2014). Efficacy of citric acid denture cleanser on the Candida albicans biofilm formed on poly (methyl methacrylate): effects on residual biofilm and recolonization process. BMC Oral Health, 14(1): 77.
- Flemming, H.C., Wingender, J., Mayer, C., Korstgens, V., Borchard, W. (2000). Cohesiveness in biofilm matrix polymers. In Symposia-Society for General Microbiology, Cambridge; Cambridge University Press, pp. 87-106.
- Flint, S.H., Bremer, P.J., Brooks, J.D. (1997). Biofilms in dairy manufacturing plant description, current concerns and methods of control. Biofouling, 11(1): 81-97.
- Ghandbari, E.H. (1983). Reactions of Chlorine and Chlorine Dioxide with Free Fatty Acids, Fatty Acid Esters, and Triglycerides. In Water Chlorination: Environmental Impact and Health Effects, Vol. 4 ((Jolley, R.L., Brungs, W.A., Cotruvo, J.A., Cumming, R.B., Mattice, J.S., Jacobs, V.A., Eds.). Ann Arbor Sci. Publ., Ann Arbor. Mich, 167-180.
- Gómez-López, V.M., Marín, A., Medina-Martínez, M.S., Gil, M.I., Allende, A. (2013). Generation of trihalomethanes with chlorine-based sanitizers and impact on microbial, nutritional and sensory quality of baby spinach. Postharvest Biol Technol., 85: 210-217.
- Granum, P.E., Lund, T. (1997). Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett, 157(2), 223-228.
- Grutsch, A.A., Nimmer, P.S., Pittsley, R.H., McKillip, J.L. (2018). Bacillus spp. as Pathogens in the Dairy Industry. Foodborne Diseases, 193-211.
- Koutina, G., Skibsted, L.H. (2015). Calcium and phosphorus equilibria during acidification of skim milk at elevated temperature. Int Dairy J., 45: 1-7.
- Kreske, A.C., Ryu, J.H., Pettigrew, C.A., Beuchat, L.R. (2006). Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm. J Food Prot, 69(11): 2621-2634.
- Lindsay, D., Brözel, V.S., Mostert, J.F., Von Holy, A. (2000). Physiology of dairy-associated Bacillus spp. over a wide pH range. Int J Food Microbiol., 54(1): 49-62.
- Lücking, G., Stoeckel, M., Atamer, Z., Hinrichs, J., Ehling-Schulz, M. (2013). Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage. Int J Food Microbiol., 166(2): 270-279.
- Marchand, S., De Block, J., De Jonghe, V., Coorevits, A., Heyndrickx, M., Herman, L. (2012). Biofilm formation in milk production and processing environments; influence on milk quality and safety. Comp Rev Food Sci Food Safety, 11(2): 133-147.
- Mittelman, M. W. (1998). Structure and functional characteristics of bacterial biofilms in fluid processing operations. J Dairy Sci., 8(10), 2760-2764.
- Neal-McKinney, J.M., Lu, X., Duong, T., Larson, C.L., Call, D.R., Shah, D.H., Konkel, M.E. (2012). Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLoS One, 7(9): e43928.
- Noss, C.I., Dennis, W.H., Olivieri, V.P., 1983. Reactivity of Chlorine Dioxide with Nucleic-acids and Proteins. In Water Chlorination: Environmental Impact and Health Effects, Vol. 4 ((Jolley, R.L., Brungs, W.A., Cotruvo, J.A., Cumming, R.B., Mattice, J.S., Jacobs, V.A., Eds.). Ann Arbor Sci. Publ., Ann Arbor. Mich, 1077–1086.
- Ntrouka, V., Hoogenkamp, M., Zaura, E., van der Weijden, F. (2011). The effect of chemotherapeutic agents on titanium‐adherent biofilms. Clin Oral Implants, 22(11): 1227-1234.
- Organji, R.S., Abulreesh, H.H., Elbanna, K., Osman, G.E.H., Khider, M. (2015). Occurrence and characterization of toxigenic Bacillus cereus in food and infant feces. Asian Pac J Trop Biomed., 5(7): 515-520.
- Oulahal, N., Brice, W., Martial, A. Degraeve, P. (2008). Quantitative analysis of survival of Staphylococcus aureus or Listeria innocua on two types of surfaces: polypropylene and stainless steel in contact with different dairy products. Food Control, 19: 178-185.
- Pagedar, A., Singh, J. (2012). Influence of physiological cell stages on biofilm formation by Bacillus cereus of dairy origin. Int Dairy J., 23(1): 30-35.
- Parkar, S.G., Flint, S.H., Palmer, J.S., Brooks, J.D. (2001). Factors influencing attachment of thermophilic bacilli to stainless steel. J Appl Microbiol, 90(6): 901-908.
- Ramos-Villarroel, A.Y., Martín-Belloso, O., Soliva-Fortuny, R. (2015). Combined effects of malic acid dip and pulsed light treatments on the inactivation of Listeria innocua and Escherichia coli on fresh-cut produce. Food Control, 52: 112-118.
- Ricke S.C. (2003). Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sciences, 82: 632-639.
- Ryu, J.H., Beuchat, L.R. (2005). Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid–based sanitizer. J Food Prot, 68(12), 2614-2622.
- Sadiq, R., Rodriguez, M.J. (2004). Disinfection by-products (DBPs) in drinking water and predictive models for their occurrence: A review. Sci Total Environ. 321: 21-46.
- Sánchez, G., Elizaquível, P., Aznar, R., Selma, M.V. (2015). Virucidal effect of high power ultrasound combined with a chemical sanitizer containing peroxyacetic acid for water reconditioning in the fresh-cut industry. Food Control, 52: 126-131.
- Schaeffer, A.B., Fulton, M.D. (1933). A simplified method of staining endospores. Science, 77(1990): 194-194.
- Simões, M., Simoes, L.C., Vieira, M.J. (2010). A review of current and emergent biofilm control strategies. LWT-Food Sci Technol, 43(4): 573-583.
- Stepanović, S., Vuković, D., Dakić, I., Savić, B., Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods, 40(2): 175-179.
- Te Giffel, M.C., Beumer, R.R., Leijendekkers, S., Rombouts, F.M. (1996). Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiology, 13(1), 53-58.
- Van Haute, S., Sampers, I., Holvoet, K., Uyttendaele, M. (2013). Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing. Appl Environ Microbiol, 79(9): 2850-2861.
- Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J. (2005). Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate. Appl Environ Microbiol, 71(9): 5022-5028.
- Waters, B.W., Hung, Y.C. (2014). The effect of organic loads on stability of various chlorine‐based sanitisers. Int J Food Sci Technol, 49(3): 867-875.
- Zhang, Q.Q., Ye, K.P., Juneja, V.K., Xu, X. (2017). Response surface model for the reduction of Salmonella biofilm on stainless steel with lactic acid, ethanol, and chlorine as controlling factors. Journal of Food Safety, 37(3).1-7.