Effects Of Some Spices Extracts On Biofilms Of Three Foodborne Pathogens

Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus are the common foodborne pathogens, they all could affect the food quality and safety, and then threaten the human health. The biofilm is a community composed of microbes and the extracellular polymeric substances secreted by them. This special architecture is benefit for microbes in the biofilms surviving from the exposure of antibiotics and disinfectants. But this mechanism could bring potential serious risks in the fields of medical and food safety. The traditional strategy to reduce these risks is using the antibiotics, detergents and disinfectants excessively, but unfortunately, the result is limited, and it may cause bacteria to develop multiple drug resistance. Therefore, it is urgently needed to develop a safe, efficient, and natural anti-biofilm material.In this study we investigated antibacterial activities of 10 spices samples extracted via maceration by water or ethanol, then 2 spices whose antibacterial activities were more efficient were selected to assay the minimum inhibitory concentration(MIC). Following the MIC assays, the effects of extracts on the biofilms formed by three pathogens were detected using crystal violet staining method, two-fold serial dilution method, XTT reduction assays, phenol-sulfuric acid method and confocal laser scanning microscope(CLSM). The main contents and results of this study are as following:Different extracts have different actives to different strains. The garlic, cinnamon, clove, leaves and stalks of moringa oleifera lam have the stronger antibacterial activities, and Chinese prickly ash, white pepper, fennel and rosemary are lower.But there is no antibacterial activity of onion, no matter extracted by water or ethanol in this study. Generally speaking, the ethanol extracts displayed the higher inhibitory actives compared to water extracts against all tested bacteria, and the V. parahaemolyticus is more sensitive than S. aureus and E. colito spices.Compared to the inhibition zones of different spices, we selected two extracts for every strains, and assayed the MIC values. Recorded MIC values are between 6.25 mg/m L to 12.5mg/m L for all strains, respectively. The same extract showed different MIC values to different bacteria, such as garlic exhibited higher MIC values to S. aureus(12.5mg/m L)than V. parahaemolyticus(6.5mg/m L). Meanwhile, we explored the MIC values of their mixture, and regrettably, they didn’t show the stronger antibacterial activities.We explored the factors which may impact on the formation of biofilms, including p H, temperature, incubation time and nutrition. And we found that, p H have an influence on biofilm formation, but it is not accurate, in other words, the optimum p H is not a specific number, but a range, which is partial alkaline. Compared to p H, the influence of temperature is more obvious, the optimum temperature for biofilm formation of S. aureus, E. coli and V. parahaemolyticus is 37 ℃, 37 ℃ and 30 ℃,respectively. All the tested strains formed the largest biofilms after incubating 48 hours, and eutrophic medium is propitious to biofilm formation.The selected extracts were tested if they could reduce the biofilm formation of pathogens, and the results showed that, sub-MIC of all the extracts not only could inhibit the biofilm formation, but also reduce the metabolic activity and the secretion of extracellular polysaccharide of cells in the biofilms. With the aid of a CLSM, the increase of dead cells numbers and the thinning of biofilms could be observed intuitively.We also explored the abilities of extracts to eradicate the mature biofilms formed by these pathogens, and we surprisingly found that, these extracts not only could scavenge the biofilms, but also kill the bacteria in the biofilms, and reduce the attachment of polysaccharides. These results were also confirmed by the visual images obtained from CLSM.In summary, the spices not only have the antibacterial activities, but also possess the amazing activities of inhibiting and eradicating biofilms formed by foodborne pathogens.