Evaluation of acid resistance in food-associated bacteria

Abstract

Although the application of low pH is common practice in food preservation, the emergence of acid tolerance has been reported world-wide amidst a growing concern that preservation with weak acids, such as organic acids may be influenced as a result of food-borne bacteria becoming acid tolerant or acid resistant. The present study was conducted to assess the acid tolerance of a wide range of bacterial species and consequently the sustainable application of organic acids as food preservatives in particularly acidic foodstuffs. Acid tolerance was determined in 19 bacterial strains predominantly associated with food spoilage and food poisoning. After exposure to hydrochloric acid 16% of the isolates were found to be intrinsically tolerant to low pH and included amongst others the enteric bacteria Escherichia coli and Salmonella spp. The latter organisms are known causative agents in food spoilage and poisoning, and the results highlight the predicaments related to their ability to survive in acidic foodstuffs as well as the human gastric environment. Bacterial strains were further exposed to increasing concentrations of various acidic foodstuffs in order to determine the development of acid tolerance by gradual decrease in pH, as opposed to exposure to acid shock. After induction, the protein profiles of resulting acid tolerant isolates were compared with those of the original un-induced strains. Exposure to acidic foodstuffs resulted in various survival profiles, where not only pH but also the type of acidulant (foodstuff or inorganic acid) were found to be contributing factors in acid tolerance development. Bacterial protein composition after exposure to acidic foodstuffs showed considerable variation which may be indicative of acid tolerance development whereas the mechanisms involved may be the result of multiple modifications in bacterial composition.

After the induction of acid tolerance, susceptibility of induced strains to various organic acids were determined at various pH values. This was done to investigate whether acid tolerance would influence the inhibitory activity of organic acids as antimicrobial agents in acidic food. Decreased susceptibility was not significantly demonstrated with the exception of only selected isolates, the latter including E. coli and S. typhimurium. Organic acid activity was found to be much more effective at lower pH values and it would be necessary to elucidate whether this inhibition is the result of a lower pH or more specifically the activity of the organic acids. The effect of exposure to an acidic environment on phenotypic characteristics of Gram-negative bacteria, and more specifically psychrotrophic organisms was evaluated in order to show the combined effect of organic acids and low temperature preservation. The characteristic yellow pigment of various Chryseobacterium species was found to be not as apparent after acid exposure while in some cases the colonies were observed as white. In Pseudomonas aeruginosa the characteristic green pigment was much more prominent after acid exposure. These morphological alterations may be important factors that should be considered in identification procedures employed in food safety laboratories. Finally, the influence of acidic exposure via acidic foodstuffs and also organic acids on the protein composition and outer membrane protein structure of various bacterial cells was investigated. No specific relationships with the MICs (Minimum Inhibitory Concentrations) of organic acids after induction with the selected acidic foodstuffs could be established, although various differences were found in protein expression. From the results, it may be suggested that the outer membrane of various pathogenic bacteria is involved in acid tolerance development and this supports the reports on the importance of membrane integrity in the protection against low pH. In conclusion, the study endeavoured to add to the body of knowledge with regard to alternative food preservation regimes utilising organic acids, either solely or in combination with selected extrinsic and intrinsic parameters.