Plant-derived biocides for water disinfection and other hygienic applications

Abstract

The production of potentially harmful disinfection by-products and the emergence of resistant bacteria have generated interest in searching for new antimicrobial agents that are safe and ideally similarly cheap compared to existing biocides. This research aimed to investigate the inhibitory activity of extracts and individual compounds derived from banana peel, a major food waste in many parts of the world, on bacteria (Staphylococcus aureus NCTC 6571, Escherichia coli NCTC 12241) and viruses (represented by a viral surrogate, MS2 coliphage). To better understand the antimicrobial activity, generation of hydrogen peroxide, one of the potential antimicrobial mechanisms involved, was also studied. In the first part of this research, the relationship between extract composition and antimicrobial activity was studied. Banana peels subjected to one of two drying processes, freeze drying and oven drying, were extracted at different maceration times and the antimicrobial activity of the extracts was determined using a broth micro-dilution method. Freeze-dried banana peels extracts from three hours of maceration were found to be the most active for both bacteria tested. Following this, compound identification was carried out on the freeze-dried banana extracts using high-performance liquid chromatography (HPLC) to determine the active compounds that are responsible for the extracts’ antimicrobial activity. Three compounds, namely gallic acid, catechin and epicatechin, were found to be present in all extracts and therefore were chosen for further study individually. Epigallocatechin gallate (EGCg), a related catechin that has already been studied by others previously, was also included. To determine the relative efficacy of the extracts and their individual compounds as biocides, a disinfection experiment was carried out. The results showed that higher inhibitory activity by the extracts was observed in S. aureus than E. coli. Among all individual compounds tested, EGCg was shown to have the most potential to be explored as a biocide, meanwhile only relatively weak activity (i.e. less than 0.5-log reduction) was exhibited by the banana peel-derived compounds. Nevertheless, the combination of two banana peel-derived antimicrobial compounds was shown to improve the inhibitory activity – i.e. higher S. aureus reduction was achieved compared to the sum of the reduction achieved when the compounds were tested individually, though no synergy between the compounds was observed for E. coli. Using a plaque assay, possible antiviral activity of the extracts and individual compounds against MS2 coliphage was determined. The results showed that MS2 was highly resistant to the extracts and all compounds tested, suggesting that banana peel extracts and their individual compounds may not be suitable to be used as virucides. Previously the inhibitory activity of these compounds had been suggested to be potentially due to the generation of hydrogen peroxide. Therefore, in this study the amounts of hydrogen peroxide generated were quantified using the ferrous oxidation-xylenol orange (FOX) assay. The results showed that micro-molar concentrations of hydrogen peroxide were indeed generated under the conditions of the disinfection experiments. This value however is too low for significant reduction in bacterial viability to be observed, and instead it was likely that the inhibitory action was mainly due to other mechanisms, such as binding to vital components in the cells. The results also indicate that the inhibitory action of EGCg treatment was not due to hydrogen peroxide generation, but likely due to direct interaction of the molecule to the peptidoglycan with cells, as proposed by other researchers. Overall, this study was the first to evaluate the efficacy of natural biocides derived directly from a waste material, against a range of target microorganisms. The results of this study demonstrate the potential of some banana peel-derived individual compounds against some species of bacteria, and demonstrated that there are synergies between two of the compounds in particular. Potential future commercialisation of such compounds as biocides (e.g. EGCg shows the most promise) would require further optimisation of these synergies and a method for cost-effectively scaling up the extraction procedures that are presented in this thesis.