Abstract
Honey is a food with high popularity, consumption and availability. The effective antibacterial activity of honey, without any reported bacterial resistance or side effects, increases its use in traditional medicine for treating many different kinds of infections. Initially, this study examined the antibacterial activity of honey against Campylobacter jejuni. Six different types of honey, which were commercially available in the UK, Saudi Arabia and New Zealand, were assessed. Honey samples were introduced to Muller Hinton media in three different ways: filtration, autoclaving honey alone and autoclaving honey with media. C. jejuni NCTC 11168 showed good sensitivity to all six investigated honey samples. The minimum inhibitory concentrations ranged between 2 and 10 % for filtered honey. Thermal treatment increased the activity of some honey types and the MIC was 4% for all honey samples, except for clear honey (C. ) Heating honey (2 %, v/v) with the medium resulted in an increase in antibacterial activity with C. jejuni, reducing to undetectable levels after 6 hours of incubation. A similar result was observed when honey was mixed with casein, followed by thermal treatment. This result indicates that thermal treatment generates a selection of new antibacterial agents in the honey-casein mixture (honey-casein MRPs). Factors including temperature and the type of sugar and amino acid present were shown to be important in the generation of antibacterial activity. Heating honey, but not other sugars, with casein or some of its constituent amino acids (e. g. lysine) at 121 C for 15 minutes was shown to be the optimal condition. Further tests revealed that the mixture was effective in vivo and in vitro. A dramatic decrease in C. jejuni colonies to below the limit of detection was observed after 6 hours of incubation in UHT milk with 2 % honey-casein MRPs. The antibacterial activity was also confirmed in an animal model, and C. jejuni was not able to cause infection in Galleria mellonella larvae in the presence of 2% honey-casein MRPs. A significant reduction in the number of C. jejuni cells invading Caco-2 cells was observed when honey-casein MRPs were added to the medium. It is considered that after the treatment (with honey-casein MRPs), C. jejuni was viable, but they lost their culturability and entered a VBNC state according to LIVE/DEAD BacLight staining. The state was irreversible and C. jejuni was not able to recover after transferring to nutrient-rich media. The exposure of C. jejuni to honey-casein MRPs also changed the microbial cell morphology from spiral to coccoid shaped, as confirmed by TEM images. The mixture was also examined against other bacteria such as E. coli, S. typhimurium, L. monocytogenes, S. aureus and H. pylori; but, interestingly, the effects were more limited. A high-performance liquid chromatography technique was used to assess the mixture (honey-casein MRPs). Developing the method for Maillard reaction products separation was successful and a higher number of peaks were observed. This study, overall, has highlighted the importance of honey as an antibacterial agent, as well as a source of other useful compounds formed from honey and specific amino acids under appropriate conditions. These new agents may potentially be used as additives in a new strategy to improve food safety and reduce Campylobacter prevalence in foods.