A novel approach for controlling foodborne pathogens using modified atmosphere and Lactobacillus reuteri DPC16 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Albany, New Zealand

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The current trend of increasing demand for minimally processed food requires more effective preservation technologies than are presently used. In this study, an investigation has been made into a novel strategy to control some common foodborne pathogens, and therefore, to provide an alternative means for enhancing the safety and extending the shelf lives of food products. Modified atmosphere is able to extend the shelf life of seafood and meat products. In this study, a simulated controlled atmosphere (CA) broth system was used to investigate the potential of a modified atmosphere rich in CO2 at a concentration of 40%, supplemented with N2, to control common foodborne pathogens, such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus. Controlled atmosphere significantly reduced the exponential growth rates of all tested pathogens, while the effects on other growth parameters (eg. lag phase duration and maximum population density) depended on the individual species and the specific growth conditions. The CA significantly extended the lag phase durations of S. aureus and V. parahaemolyticus at 20 degrees C at both pH 6.3 and 6.8, and that of L. monocytogenes at both 7 degrees C and 20 degrees C, and at both pH 6.3 and 6.8. The CA also significantly lowered the maximum population densities of S. aureus and V. parahaemolyticus at 20 degrees C, at pH 6.3 and 6.8, S. Typhimurium at pH 6.8, and L. monocytogenes at pH 6.3 and 7 degrees C. E. coli O157:H7 and S. Typhimurium were more resistant to the inhibitory effect of the CA, while S. aureus and V. parahaemolyticus were most sensitive. The inhibitory effect of CA was due mainly to the extensions of the lag phase duration and the reduction of the exponential growth rates of the test pathogens. This study confirms other studies that CA as a means for food preservation provides potential to control foodborne pathogens and therefore enhance the safety of a food product. The use of lactic acid bacteria (LAB) in controlling spoilage microorganisms and pathogens in foods has been a popular research theme worldwide. In this study, the antimicrobial effects of 18 lactic acid bacteria strains were evaluated in vitro, with emphasis on the most effective strain, the newly characterised Lactobacillus reuteri DPC16. The results demonstrated antagonistic effects of many strains against L. monocytogenes, E. coli O157:H7, S. Typhimurium and S. aureus. L. reuteri DPC16 showed the strongest antimicrobial activity against the tested pathogens including both Gram-positive and Gram-negative bacteria. Co-cultivation of L. reuteri DPC16, and co-incubation of its spent culture supernatant (DPC16-SCS), with the pathogens have demonstrated that the antimicrobial effect is bactericidal and valid at pH 4 - 6.5 and at a temperature as low as 10 degrees C. Further characterisation of the antimicrobial effect of L. reuteri DPC16 showed it to be mainly due to the presence of reuterin (ß-hydroxypropionaldehyde), although lactic acid may have also played a role. These characteristics of L. reuteri DPC16 and its metabolite reuterin make it an unique and potent candidate as a biopreservative to control both Gram-positive and Gram-negative bacteria in foods. The combination of L. reuteri DPC16 and CA was assessed for its inhibitory effect on L. monocytogenes using DPC16-SCS and the fermentative supernatant of L. reuteri DPC16 from a glycerol-water solution (DPC16-GFS). The results showed that both of these supernatants, at 25 AU/mL, in combination with CA (60% CO2:40% N2) had a combined inhibitory effect on L. monocytogenes which could not be achieved by any one of the individual factors alone. Analysis of the levels of expression of some stress response genes of L. monocytogenes, after growth in the presence of L. reuteri DPC16 supernatant and/or CA, showed that the expression of some genes was affected including genes betL, gbuA and opuCA responsible for osmosis adaptation and genes gadA, gadB and gadC responsible for acid tolerance. Induction of gbuA, gadB and gadC by the culture supernatant suggests activation of osmotic and acid adaptation and that these genes play a major role in the culture supernatant-induced stresses. An investigation was also carried out to determine if the changes in gene expression conferred a cross-protection to heat. The result showed that the survival of L. monocytogenes grown in the presence of the culture supernatant and CA was significantly increased after exposure to heat treatment at 56oC, suggesting that a cross-protection to thermal stress had been induced. Based on these findings it is proposed that a comprehensive novel strategy incorporating both L. reuteri DPC16 or its fermentative products and a modified atmosphere rich in CO2 could be developed to potentially control foodborne pathogens in food products.
Lactobacillus reuteri, Microbiology, Protective atmospheres, Food preservation