Antimicrobial activity of citric acid against Escherichia coli, Staphylococcus aureus and Candida albicans as a sanitizer agent

DOI: 10.3195/ejejfs.787021 Eurasian Journal of Forest Science 2020 8(3): 295-301 http://dergipark.gov.tr/ejejfs Antimicrobial activity of citric acid against Escherichia coli, Staphylococcus aureus and Candida albicans as a sanitizer agent Elif Ayşe Erdoğan Eliuz Mersin University, Technical Sciences Vocational School, Department of Food Technology, TR-33343, Mersin, Turkey Corresponding author: Abstract In this study, it was aimed to investigate the antimicrobial activity and inhibition percentage of citric acid against E. coli, S. aureus and C. albicans as a sanitizer agent. It was researched that persistent/tolerant levels of microorganisms in the citric acid presence. Spectrophotometric Broth Microdilution Method and Surface Disinfection Test were used for antimicrobial activity and Inhibition percentage, respectively. Modified TDtest was used for persistent/tolerant levels of microorganisms. While MIC (Minimum Inhibitory Concentration) of citric acid on E. coli and S. aureus were 0.06 g/mL, it was 0.03 g/mL for C. albicans. It was applied the TDtest with citric acid solution and discriminate by tolerance level of E. coli than the other microorganisms at the end of the 48-h incubation. In a final test which was investigated the survival of E. coli, S. aureus and C. albicans exposure to citric acid by Surface Disinfection Test, there was no statistically significant difference between bacteria and yeast in terms of the number of inhibited cells. Although citric acid was rather active against all three microorganisms, the strongest and permanent antimicrobial effect was on C. albicans. Finally, citric acid should be used especially against anti-Candida albicans in permanent hygiene practices. Keywords: Citric acid, E. coli, S. aureus, C. albicans, Persistent/tolerant, Sanitizer Introduction Citric acid known as tricarboxylic acid (C6H8O7) is an preservative, acidulate, flavouring, emulsifier, sequestrate and buffering agent widely used across many industries such as food, beverage, pharmaceutical and cosmetic products (Thauer et al. 1988, Verhoff 2005). It is naturally found in Lime, Lemon, Raspberry, Tomato, Pineapple, strawberry, cranberry and was first crystallized from lemon juice and named by Scheele in Sweden in 1784 (Scheele 1784, Ciriminna et al. 2017). Colourless and odourless citric acid is slightly hygroscopic and highly soluble in water (62.07% at 25°C) and slightly hygroscopic (Dalman 1937). Its environmentally benign nature and quickly degrades in surface waters besides useful Physico-chemical structure make it ideally suited for food, cosmetic and pharmaceutical applications. The code of citric acid is E330 as a food ingredient in European Union (E331 and E332, respectively, for sodium and potassium citrate) and has been Generally Recognized as Safe (GRAS) in the US (Ciriminna et al. 2017). Citric acid's popularity has increased even more in household detergents and home hygiene because of restrictions on the use of phosphate in dishwasher detergents in the US (since 2010), EU (Ciriminna et al. 2017) and Turkey (Official Newspaper 2018).This situation has caused the citric acid market to take more place in the industrial field and will follow. It was shown that citric acid has an excellent antiviral disinfectant effect and reported to irreversibly disturb the virus's 295 Eurasian Journal of Forest Science – Antimicrobial activity of citric acid by Erdoğan Elius 2020 structure. For this reason, it has been suggested that it can be used for permanent disinfection on the infected surfaces (Koromyslova et al. 2015). It is industrially added to juices in the beverage industry and is widely used to increase the stability of concentrates. At the same time, thanks to its antioxidant and antimicrobial properties against foodborne pathogens such as Escherichia coli, Salmonella, Listeria, monocytogenes., it prevents spoilage and keeps the drink fresh (Rossle et al. 2009; Ciriminna et al. 2017). Besides, it is stated that will be needed more biotechnological products such as citric acid which do not have serious harm to nature and human, and which have useful powerful effects, in the future (Ciriminna et al. 2016). Insufficient hygiene of food processing products and surfaces contributes to long-term surface trapping of pathogen microorganisms, cross-contamination and irreversible food contamination (Carpentier and Cerf 1993). Therefore, surfaces often are disinfected with many active chemicals, but most of the chemicals pose a concern for food safety as they leave residue and pose a threat to health (Akinyemi et al. 2006). Therefore, the importance of naturally originated antimicrobials is increasing day by day (Nural et al. 2018; Yabalak et al. 2020). Besides, microorganisms' gaining resistance against these chemicals over time makes more difficult achieve sufficient and permanent hygiene (Notermans et al. 1991). In the present paper, it was first investigated the tolerance or persistence levels in E. coli, S. aureus and C. albicans exposed to the solution according to the modified Kirby–Bauer disk diffusion method besides evaluating the antimicrobial actitivity of citric acid as a sanitizer agent. Materials and Methods Materials and instruments Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), Triptic Soy Broth (TSB), Sabouraud Dextrose Broth (SDB), were supplied from Merck (Germany). E. coli (ATCC 25293), S. aureus and C. albicans were taken from Refik Saydam Hıfzıssıhha Centre (Turkey). ELIZA spectrophotometer (MULTISKAN GO, Thermo Scientific) was used for antimicrobial measurements. Citric acid was purchased from Sigma. Preparing the citric acid solution To prepare the citric acid solution, 1 g citric acid was weighed and added to 30 mL of previously sterilized distilled water. It was then homogenized in the magnetic stirrer for 1 hour and stored at 4 oC. The pH value of citric acid in water was determined using Ph meters. Antimicrobial screening The inoculums of Escherichia coli (ATCC 25293), Staphylococcus aureus (ATCC 25923) and Candida albicans (ATCC 90028) were prepared in 4 mL TSB for bacteria and 4 mL SDB for yeast and incubated at 37oC, overnight. After 24 hours, the microorganism suspensions were adjusted to 0.5 McFarland Standard Turbidity and stored at +4 °C until experiments. Broth Microdilution Method The 50 µL of MHB medium were added into 96-well microtiter plates and two-fold serial dilutions of 50 µL citric acid solution were made x-axis along from 2nd to 10th columns and used columns 11 and 12 as the negative control (only MHB and microbe). Then, 5 μL cultures were inoculated on all wells except negative control. As positive controls, Ampicillin (AMP: 128 µg/mL) in bacteria and Fluconazole (FLC: 128 µg/mL) in yeast were used. Finally, all plates were incubated at 37oC for 24 hours and the growth (turbidity) was measured at 615 nm. MIC was determined as the lowest concentration where no visible turbidity was observed in the eac (...truncated)