title: Tetramethylbenzidine method for monitoring the free available chlorine and microbicidal activity of chlorite-based sanitizers under organic-matter-rich environments.
abstract: Chlorine is a principal disinfectant for food and environmental sanitation. Monitoring of free available chlorine (FAC) is essential for ensuring the efficacy of food disinfection processes that rely on chlorine. N,N-diethyl-p-phenylenediamine (DPD) is commonly used for FAC monitoring. However, here, we show that upon contact with bovine serum albumin (BSA) or broiler carcasses, chlorite (HClO2 )-based sanitizers acquire a pink colour, which can interfere with measurement of oxidized DPD absorbance at 513-550 nm. Alternatively, the pink colour did not interfere with 3,3',5,5'-tetramethylbenzidine (TMB)-based FAC monitoring. The FAC levels of NaClO and weakly acidified chlorous acid water (WACAW) were first adjusted by the TMB method and the killing activity of these sanitizers towards methicillin-resistant Staphylococcus aureus (MRSA) and feline calicivirus (FCV) was compared in the presence or absence of 0·5% BSA. At 200 ppm FAC, NaClO lost its bactericidal activity against MRSA after 10-min incubation with 0·5% BSA. Meanwhile, under the same conditions WACAW reduced the number of bacteria to below the detection limit. Similar results were obtained with FCV, indicating that the chlorite-based WACAW sanitizer is relatively stable under organic-matter-rich conditions. Moreover, TMB is suitable for in situ FAC monitoring of chlorite-based sanitizers in food and environmental disinfection processes.
  
  
  CONCLUSIONS
  For practical applications of chlorine in food processing, monitoring of FAC is critical to validate disinfection efficacy. In this study we found that chlorite-based sanitizers acquired a pink colour upon contact with BSA or broiler carcasses. This pink colour interfered with FAC monitoring by methods that measure oxidized N,N-diethyl-p-phenylenediamine absorbance between 513-550 nm. Alternatively, FAC levels of chlorite-based sanitizers could be monitored using the absorbance of 3,3',5,5'-tetramethylbenzidine at 650 nm, which does not overlap with the acquired pink colour. These data provide valuable information for safety management of disinfection processes that use chlorite-based sanitizers.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, Kagawa, Japan.|Honbu Sankei Co. Ltd., Chuou-ku, Osaka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamaoka
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nakayama-Imaohji
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, Kagawa, Japan.|Honbu Sankei Co. Ltd., Chuou-ku, Osaka, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Horiuchi
authors.affiliation: Department of Cellular and Molecular Medicine, Wakayama Medical University Graduate School of Medicine, Wakayama, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamasaki
authors.affiliation: Faculty of Nursing, Shikoku University, Tokushima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nagao
authors.affiliation: Honbu Sankei Co. Ltd., Chuou-ku, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Fujita
authors.affiliation: Faculty of Fisheries, Kagoshima University, Kagoshima-shi, Kagoshima, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.affiliation: Honbu Sankei Co. Ltd., Chuou-ku, Osaka, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Goda
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kuwahara
mesh_terms: D000818:Animals; D001560:Benzidines / Q000737:chemistry*; D017927:Calicivirus, Feline / Q000187:drug effects*; D002645:Chickens; D002712:Chlorides / Q000737:chemistry / Q000494:pharmacology*; D002713:Chlorine / Q000032:analysis*; D015169:Colony Count, Microbial; D004202:Disinfectants / Q000494:pharmacology*; D004203:Disinfection / Q000379:methods; D005511:Food Handling / Q000379:methods; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D010655:Phenylenediamines / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D014867:Water
chemical_list: D001560:Benzidines; D002712:Chlorides; D004202:Disinfectants; D010655:Phenylenediamines; D014867:Water; D012710:Serum Albumin, Bovine; C021758:3,3',5,5'-tetramethylbenzidine; D002713:Chlorine; C063160:chlorous acid; C013935:N,N-diethyl 4-phenylenediamine; C001599:chlorite
keywords: 3,3′,5,5′-tetramethylbenzidine; N,N-diethyl-p-phenylenediamine; bacteria; bovine serum albumin; chlorous acid water; feline calicivirus; free chlorine; methicillin-resistant Staphylococcus aureus