The Application of Spices and Herbs to Control Food-borne Pathogens in Ready to Eat Shrimp

Abstract

Abstract An interest in antimicrobials from new sources has expanded in recent years because of greater consumer demand for healthy foods with natural preservatives instead of chemical preservatives. Undetermined antimicrobial and antioxidant properties of novel or less-utilised spices and herbs may have the potential to fulfil consumer demands. Numerous studies have reported the antimicrobial activity of individual essential oils or plant extracts alone, however only a few have reported on the activity of combined spice and herb extracts. In addition, there is lack of knowledge on the mechanism of antimicrobial activity of spice and herb extracts. The overall aim of this study was to investigate the antimicrobial activity of less well known spices and herbs against food-borne pathogens Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, Clostridium perfringens and Staphylococcus aureus. In total, seven herbs and spices including the less-utilized goraka (Garcinia quaesita), galangal (Alpinia galanga), lemon iron bark (Eucalyptus staigerana) and mountain pepper (Tasmannia lanceolata) and the more commonly used pepper (Piper nigrum), rosemary (Rosmarinus officinalis), and oregano (Oreganum vulgare) were extracted using ethanol, water and hexane solvents. The antimicrobial activities of these extracts were tested using disc diffusion and broth dilution assays. The total phenolic content of spice and herb extracts was assayed to establish any relationship between antimicrobial activity and phenolic compound levels. Solvent type greatly influenced the antimicrobial activity of the spice and herb extracts except for those of pepper, which had little or no activity. In particular, the hexane and ethanol extracts of galangal and ethanol and water extracts of lemon iron bark had strong antimicrobial activity against S. aureus and/or L. monocytogenes. Antimicrobial activity and phenolic compound levels was found to poorly correlated (r2< 0.30), in contrast to that found by others. This indicated that the antimicrobial activity in some herb and spice extracts may be due to the presence of substances other than phenolic compounds. Three spice and herb extracts yielding the best results were selected for synergistic antimicrobial activity studies using broth dilution assay followed by viable counts. The chemical compositions of these extracts were determined using gas chromatography-mass spectrometry (GC-MS) analysis to provide further insight into antimicrobial constituents and their potential mechanism(s) of action. Combinations of galangal with either rosemary or lemon iron bark showed synergistic antimicrobial activity. Specifically, galangal and rosemary showed synergistic activity against S. aureus and L. monocytogenes only, while galangal and lemon iron bark showed synergistic activity against E. coli and S. Typhimurium. The major chemical components of the galangal and lemon iron bark extracts were 1'-acetoxy-chavicol acetate (1'ACA) (63.4%), and neral (15.6%), respectively, while 1,8 cineole (26.3%) and camphor (20.3%) were identified as major chemical components of the rosemary extract. Previously works by others have shown that crude galangal extract causes cell leakage and coagulation of cytoplasmic components. This part of the project focused on determining if 1'ACA was the galangal extract component which caused membrane damage and if so what effects it has on cell morphology, membrane fatty acid composition, stress response and virulence gene expression and mutation rate. The major chemical compound of the galangal extract (1'ACA) was purified to 100% and was shown to possess antimicrobial activity against S. aureus (including methicillin resistant strains) and L. monocytogenes, but little activity against E. coli and S. Typhimurium. The mechanism of action of purified 1'ACA against the most sensitive bacteria was investigated using different methods. Scanning electron microscopy showed that 1'ACA caused S. aureus cells to develop a rough surface and appear damaged or deformed. An increase in levels of cytoplasmic constituent release and a higher molar fatty acid unsaturation index (due to the changes of ratio of saturated to unsaturated fatty acids) provided further evidence of the effect of 1'ACA on cell envelope integrity. Significant up regulation of genes responsive to cell wall (msrA, vraS) and membrane damage (vraD) were identified using reverse-transcriptase quantitative polymerase chain reaction. Furthermore, a significantly higher mutation rate was observed in S. aureus exposed to 1'ACA. The results of this study suggest that the S. aureus cell envelope is the principal target for the microbicidal action of 1'ACA, either directly or indirectly. Lastly the use of synergistic herb and spice extract combinations in controlling pathogens and food spoilage bacteria in vacuum packed ready-to-eat (RTE) shrimp was investigated. A combination of galangal, rosemary and lemon iron bark significantly (