Microbiological hygiene and biological control of leafy green vegetables

Abstract: Fruit and vegetables are vital components of a healthy diet, and international strategies to encourage their consumption are in place (FAO/WHO, 2005). Ready-to-eat (RTE) leafy green vegetable products have exploded in popularity, and are a convenient and attractive way to add greens to the plate. Unfortunately, sales numbers are accompanied by increasing numbers of food-borne illness outbreaks.This thesis focuses on the microbial hygiene from a consumer’s perspective, followed by application and evaluation of biological control as a mean of reducing the risk of food-borne illness. When studying the efficacy of two different household washing methods to reduce the bacterial load on leafy green vegetables, it was seen that only after rinsing at high water velocity (8 L/min), after five repetitions, the bacterial load decreased with 90 %. The treatment disintegrated the leaves, and still left the produce with high amounts of culturable bacteria. These results highlight the inefficiency of tap water washing methods available for the consumer. Consumer habits are also important to consider when assessing the microbial hygiene of food products. Packages of RTE leafy green products were opened, stored at 7 ˚C and compared with unopened bags. The total aerobic count from different producers varied greatly and no correlation to opened bags could be made. Neither could bacterial levels be linked to a certain producer or product type.Inoculation with E. coli strains indicate that the type strain is able to survive, but not multiply, in household conditions. However, wild strains of E. coli were seen to multiply at different time-points during the shelf-life period, adapting to cold storage conditions. This varying and unpredictable bacterial status of ready to eat leafy green products calls for new strategies to reduce unwanted microorganisms and prevent food-borne illness.By the means of biological control, bacteria can be used to counteract food safety hazards. Therefore, isolates antagonistic to E. coli have been isolated and evaluated in an industrial field production setting. Selected isolates showing antagonism in vitro were coated onto spinach seeds before planting. Next generation sequencing analysis revealed that the microbiota of the plants inoculated with the selected strains was altered in a beneficial direction, and a reduction of Escherichia-Shigella could be seen during the development from seed to plant.As a tentative safety evaluation of the selected strains for biological control, an individual comparison for immunomodulating effects in mice was made. The two Bacillus coagulans strains consistently resemble the response of untreated animals, which must be considered a positive trait. The strain of Pseudomonas punonensis had a weaker influence on the immune system, while the Pseudomonas cedrina strain and the Rhocococcus cerastii strain induced inflammatory responses. The P. punonensis strain and one B. coagulans strain increased the microbiota diversity, which is correlated to host health.These results encourage the usage of bacterial antagonists as part of the solution to reduce the risk of human pathogens on leafy green vegetables.