Article Search
닫기

Microbiology and Biotechnology Letters

보문(Article)

View PDF

Food Microbiology (FM)  |  Food Borne Pathogens and Food Safety

Microbiol. Biotechnol. Lett.

Received: April 27, 2022; Revised: May 26, 2022; Accepted: May 31, 2022

산성 pH 조건에서 차아염소산나트륨의 항균 활성 향상

Enhanced Antibacterial Activity of Sodium Hypochlorite under Acidic pH Condition

Hyeon-Bin Son, Won-Bin Bae, and Kwang-Hwan Jhee*

Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi 39177, Republic of Korea

Correspondence to :
Kwang-Hwan Jhee,       khjhee@kumoh.ac.kr

Sodium hypochlorite (NaClO) is a disinfectant widely used in hospitals and food industries because of its antimicrobial activity against not only bacteria but also fungi and virus. The antibacterial activity of NaClO lies in the maintenance of a stable hypochlorous acid (HClO) concentration, which is regulated by pH of the solution. HClO can easily penetrate bacterial cell membrane due to its chemical neutrality and the antibacterial activity of NaClO is thought to depend on the concentration of HClO in solution rather than hypochlorite ions (ClO). In this study, we investigated the antibacterial activity of NaClO according to pH adjustment by means of time kill test and assays of Reactive Oxygen Species (ROS) and adenosine triphosphate (ATP) concentration changes before and after NaClO treatment. We also investigated that the degree of cell wall destruction through field emission scanning electron microscopy (FE-SEM). Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) exposed to 5 ppm NaClO at pH 5 exhibited 99.9% mortality. ROS production at pH 5 was 48% higher than that produced at pH 7. In addition, the ATP concentration in E. coli and S. aureus exposed to pH 5 decreased by 94% and 91%, respectively. As a result of FE-SEM, it was confirmed that the cell wall was destroyed in the bacteria by exposing to pH 5 NaClO. Taken together, our results indicate that the antibacterial activity of 5 ppm NaClO can be improved simply by adjusting the pH.

Keywords: Antibacterial activity, FE-SEM, hypochlorous acid (HClO), ATP, reactive oxygen species

Graphical Abstract


  1. Periyasamy T, Asrafali S, Shanmugam M, Kim S-C. 2021. Development of sustainable and antimicrobial film based on polybenzoxazine and cellulose. Int. J. Biol. Macromol. 170: 664-673.
    Pubmed
  2. Song J-H. 2011. Antimicrobial resistance in Gram-positive cocci: Past 50 years, present and future. Infect. Chemother. 43:443-449.
  3. Kerkaert B, Mestdagh F, Cucu T, Aedo PR, Ling SY, De Meulenaer B. 2011. Hypochlorous and peracetic acid induced oxidation of dairy proteins. J. Agric. Food Chem. 59: 907-914.
    Pubmed
  4. Coates D. 1985. A comparison of sodium hypochlorite and sodium dichloroisocyanurate products. J. Hosp. Infect. 6: 31-40.
  5. Topić F, Marrett JM, Borchers TH, Titi HM, Barrett CJ, Friščić T. 2021. After 200 years: The structure of bleach and characterization of hypohalite ions by single-crystal X-ray diffraction. Angew. Chem. Int. Ed. Engl. 60: 24400-24405.
    Pubmed
  6. Lee D, Howlett J, Pratten J, Mordan N, McDonald A, Wilson M, et al. 2009. Susceptibility of MRSA biofilms to denture-cleansing agents. FEMS Microbiol. Lett. 291: 241-246.
    Pubmed
  7. Gerritsen CM, Margerum DW. 1990. Non-metal redox kinetics:hypochlorite and hypochlorous acid reactions with cyanide. Inorg. Chem. 29: 2757-2762.
  8. Wang TX, Margerum DW. 1994. Kinetics of reversible chlorine hydrolysis: temperature dependence and general-acid/baseassisted mechanisms. Inorg. Chem. 33: 1050-1055.
  9. Wang L, Bassiri M, Najafi R, Najafi K, Yang J, Khosrovi B, et al. 2007. Hypochlorous acid as a potential wound care agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. J. Burns Wounds. 6: e5.
  10. Fukuzaki S. 2006. Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol Sci. 11: 147157.
    Pubmed
  11. Severing A-L, Rembe J-D, Koester V, Stuermer EK. 2018. Safety and efficacy profiles of different commercial sodium hypochlorite/hypochlorous acid solutions (NaClO/HClO): antimicrobial efficacy, cytotoxic impact and physicochemical parameters in vitro. J. Antimicrob. Chemother. 74: 365-372.
    Pubmed
  12. Dukan S, Belkin S, Touati D. 1999. Reactive oxygen species are partially involved in the bacteriocidal action of hypochlorous acid. Arch. Biochem. Biophys. 367: 311-316.
    Pubmed
  13. da Cruz Nizer WS, Inkovskiy V, Overhage J. 2020. Surviving reactive chlorine stress: responses of Gram-negative bacteria to hypochlorous acid. Microorganisms 8: 1220.
    Pubmed KoreaMed
  14. Castillo DM, Castillo Y, Delgadillo NA, Neuta Y, Jola J, Calderón JL, et al. 2015. Viability and effects on bacterial proteins by oral rinses with hypochlorous acid as active ingredient. Braz. Dent. J. 26: 519-524.
    Pubmed
  15. Spickett CM, Jerlich A, Panasenko OM, Arnhold J, Pitt AR, Stelmaszyńska T, et al. 2000. The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids. Acta Biochim. Pol. 47: 889-899.
  16. Dever G, Wainwright CL, Kennedy S, Spickett CM. 2006. Fatty acid and phospholipid chlorohydrins cause cell stress and endothelial adhesion. Acta Biochim. Pol. 53: 761-768.
    Pubmed
  17. Kim NH, Park TH, Rhee MS. 2014. Enhanced bactericidal action of acidified sodium chlorite caused by the saturation of reactants. J. Appl. Microbiol. 116: 1447-1457.
    Pubmed
  18. Feldman C, Anderson R, Kanthakumar K, Vargas A, Cole PJ, Wilson R. 1994. Oxidant-mediated ciliary dysfunction in human respiratory epithelium. Free Radic. Biol. Med. 17: 1-10.
  19. Kim HJ, Lee J-G, Kang JW, Cho H-J, Kim HS, Byeon HK, et al. 2008. Effects of a low Concentration hypochlorous acid nasal irrigation solution on bacteria, fungi, and virus. Laryngoscope 118: 1862-1867.
    Pubmed
  20. Moberg L, Karlberg B. 2000. An improved N,N'-diethyl-pphenylenediamine (DPD) method for the determination of free chlorine based on multiple wavelength detection. Anal. Chim. Acta 407: 127-133.
  21. Foerster S, Unemo M, Hathaway LJ, Low N, Althaus CL. 2016. Time-kill curve analysis and pharmacodynamic modelling for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae. BMC Microbiol. 16: 216.
    Pubmed KoreaMed
  22. Castro-Alférez M, Polo-López MI, Fernández-Ibáñez P. 2016. Intracellular mechanisms of solar water disinfection. Sci. Rep. 6:38145.
    Pubmed KoreaMed
  23. Dasgupta N, Ramalingam C. 2016. Silver nanoparticle antimicrobial activity explained by membrane rupture and reactive oxygen generation. Environ. Chem. Lett. 14: 477-485.
  24. Hong Y, Zeng J, Wang X, Drlica K, Zhao X. 2019. Post-stress bacterial cell death mediated by reactive oxygen species. Proc. Natl. Acad. Sci. USA 116: 10064-10071.
    Pubmed KoreaMed
  25. Rajneesh JP, Chatterjee A, Singh SP, Sinha RP. 2017. Detection of reactive oxygen species (ROS) in cyanobacteria using the oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA). Bio Protoc. 7: e2545.
    Pubmed KoreaMed
  26. Gomes A, Fernandes E, Lima JL. 2005. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65: 45-80.
    Pubmed
  27. Dwivedi S, Wahab R, Khan F, Mishra YK, Musarrat J, Al-Khedhairy AA. 2014. Reactive oxygen species mediated bacterial biofilm inhibition via zinc oxide nanoparticles and their statistical determination. PLoS One 9: e111289.
    Pubmed KoreaMed
  28. Solana C, Ruiz-Linares M, Baca P, Valderrama MJ, Arias-Moliz MT, Ferrer-Luque CM. 2017. Antibiofilm activity of sodium hypochlorite and alkaline tetrasodium EDTA solutions. J. Endod. 43: 2093-2096.
    Pubmed
  29. Lara HH, Ayala-Núnez NV, Turrent LdCI, Padilla CR. 2010. Bactericidal effect of silver nanoparticles against multidrug-resistant bacteria. World J. Microbiol. Biotechnol. 26: 615-621.
  30. Bhattacharya R, Saha S, Kostina O, Muravnik L, Mitra A. 2020. Replacing critical point drying with a low-cost chemical drying provides comparable surface image quality of glandular trichomes from leaves of Millingtonia hortensis L. f. in scanning electron micrograph. Appl. Microsc. 50: 15.
    Pubmed KoreaMed
  31. Fischer ER, Hansen BT, Nair V, Hoyt FH, Dorward DW. 2012. Scanning electron microscopy. Curr. Protoc. Microbiol. 25:2B.2.1-2B.2.47.
    Pubmed KoreaMed
  32. Al Shehadat S, Gorduysus MO, Hamid SSA, Abdullah NA, Samsudin AR, Ahmad A. 2018. Optimization of scanning electron microscope technique for amniotic membrane investigation:A preliminary study. Eur. J. Dent. 12: 574-578.
    Pubmed KoreaMed
  33. Romanova N, Brovko LY, Moore L, Pometun E, Savitsky A, Ugarova N, et al. 2003. Assessment of photodynamic destruction of Escherichia coli O157: H7 and Listeria monocytogenes by using ATP bioluminescence. Appl. Environ. Microbiol. 69: 63936398.
    Pubmed KoreaMed
  34. Sánchez MC, Llama-Palacios A, Marín MJ, Figuero E, León R, Blanc V, et al. 2013. Validation of ATP bioluminescence as a tool to assess antimicrobial effects of mouthrinses in an in vitro subgingival-biofilm model. Med. Oral Patol. Oral Cir. Bucal. 18:e86.
    Pubmed KoreaMed
  35. Afri M, Frimer AA, Cohen Y. 2004. Active oxygen chemistry within the liposomal bilayer: Part IV: Locating 2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrofluorescein (DCFH) and 2',7'dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer. Chem. Phys. Lipids 131: 123-133.
    Pubmed
  36. Daghastanli NA, Itri R, Baptista MS. 2008. Singlet oxygen reacts with 2',7'‐dichlorodihydrofluorescein and contributes to the formation of 2,7'‐dichlorofluorescein. Photochem. Photobiol. 84: 1238-1243.
    Pubmed
  37. Kim H, Xue X. 2020. Detection of total reactive oxygen species in adherent cells by 2',7'-Dichlorodihydrofluorescein diacetate staining. J. Vis. Exp. doi: 10.3791/60682.
    KoreaMed
  38. Borisov VB, Siletsky SA, Nastasi MR, Forte E. 2021. ROS Defense systems and terminal oxidases in bacteria. Antioxidants 10: 839.
    Pubmed KoreaMed

Starts of Metrics

Share this article on :

  • mail

Related articles in MBL

Most KeyWord ?

What is Most Keyword?

  • It is most registrated keyword in articles at this journal during for 2 years.