*3.6. Antimicrobial Activity*

Various types of honey have been tested for their antibiotic or bacteriostatic activity for a long time, increasing our knowledge of the subject. Therefore, the next purpose of this study was to enhance general knowledge about goldenrod honey's bacteriostatic (against *Escherichia coli*) and fungicidal (against budding yeast) activities using four representative samples: 4 and 8 (with typical protein and HPTLC profiles) and 3 and 4 (with distinct features). First, we compared the growth rates of both yeast and bacteria cells. As can be seen in Figure 7A, 80% and 60% honeys completely stopped the growth of microbial cells, respectively. Then we diluted the solutions of the analyzed honeys in microbiological media in the ratio of 1: 5. As shown in Figure 7B, bacterial growth was much more restricted than yeast's. The most powerful inhibitory effect on yeast was found with 80% honey solutions 4 and 8. During the first 6 h, we found no significant differences in the effect of honey on bacteria. A slightly higher bacteriostatic effect was observed in the second part of the experiment with 80% and 60% of honey 3 (Figure 7B). Then, we diluted the honeys 1:10. Compared to the untreated honey control, yeast cell growth was only slightly inhibited (Figure 7C). In turn, this dilution significantly inhibited bacterial growth, especially in honey solution 3 (Figure 7C). In summary, we observe concentration-dependent inhibition of microorganism growth, especially bacteria.

**Figure 7.** The effect of increasing doses of selected goldenrod honey samples (3, 4, 6 and 8) on budding yeast (**left**) and bacterial (**right**) growth. (**A**)—undiluted honey samples, (**B**)—samples in dilution 1:5 (200 mg/mL), (**C**)—samples in dilution 1:10 (100 mg/mL).

We then tested whether goldenrod honey and its 80% solution affected yeast viability. The dead cells were determined by fluorescently detecting the number of propidium iodidelabeled dead cells after incubation with honey and honey solution for 4 h at 28 ◦C. Figure 8 shows that the analyzed honeys do not cause 100% death of yeast cells, indicating that the arrest of growth is caused by the arrest of the cell cycle rather than the death of cells. Honey 8 displayed the highest fungicidal activity (7.67%). Meanwhile, no difference in survival

was observed between honeys 3, 4 and 6. It is interesting to note that yeast cells treated with 80% honey solution survived at a rate of 44.67%. In conclusion, goldenrod honey does not cause cell death in all yeast species. It is necessary to conduct further research to identify a yeast cell population that is resistant to honey. The age of the cell and sensitivity of young cells possibly play a role here.

**Figure 8.** The fungicidal activity of selected goldenrod honey samples (no. 3, 4, 6 and 8).

It is well known that the components of honey show different activities against various microorganisms. Honey activity is dependent on the plant species, the weather conditions and the natural properties of the nectar [34]. The use of honey as a drug for the treatment of disease dates back to 2000 BC. Aristotle first described honey as "good for sore eyes and wounds" [35]. However, only a few decades ago, the first scientific reports confirmed the antibacterial and antifungal properties of honey. It has been shown many times that honey has strong antimicrobial properties against most fungi and bacteria that cause wound and surgical infections. The microorganisms were grown in both aerobic and anaerobic conditions. As was shown, growth inhibition was complete in the media containing 100%, partial in media containing 50% and no inhibition was produced by 20% of honey. Pure honey was therefore an ideal wound dressing agent, at least on a topical basis, for surgical infections, burns and wound infections [36].

Currently available agents are no longer effective against many dermatophytes. It seems that medicine, especially dermatology, has an urgent need for new effective antifungal agents suitable for the treatment of superficial skin infections. A recent study showed that Agastache honey had fungicidal activity against dermatophytes and yeast *Candida albicans* at 40% concentration [37]. Some previously report show that honey samples from different floral sources significantly inhibit the growth of few yeast species e.g., *Candida albicans, C. krusei, C. glabrata* and *Trichosoporon* spp. Inhibition of growth tests was found to depend on the type and concentration of honey and the pathogen tested [38]. In our previous study, we reported that honeys enriched with chokeberry fruits had anti-bacterial and anti-viral properties [39]. Here, we showed that goldenrod honey has antifungal properties that significantly inhibit yeast growth, but do not result in the death of the entire population. There are likely to be two factors contributing to this: the hydration of the honey and the goldenrod flowering in autumn. It is possible that some species of bacteria and yeast may ferment this honey because it has no antiseptic properties. Apparently, these data are crucial for beekeepers, for example, in the context of honey dehydration or honey fermentation in hives.
