Experimental Design

The experimental study was conducted to determine the environmental conditions allowing growth or survival of Vibrio strains in table olives. The study was conducted consisting of two well-differentiated experiments: the first experiment was carried out in culture media (nutrient broth for Vibrio and olive brines) in Bioscreen C (Labsystems, Barcelona) so as to obtain growth curves at different parameter combinations, and the second experiment involved inoculation of pathogen in samples of table olives, which is intended to know how the behavior of Vibrio in table olives varies over time (22 days) and to study its relationship with lactic acid bacteria (LAB).

The first part of the study was carried out in a specific enrichment broth for the growth of Vibrio spp. at different pH levels (4, 5, 6, 7, 8, and 9) and NaCl concentrations (2, 4, 6, 8, 10, and 12%), making a full factorial design with a total of 36 conditions, with 10 replications each and all repeated three times. The microtiter wells were filled with 340 μl of modified culture medium. It should be taken into account that the enrichment broth used, alkaline saline peptone water (ASPW), contains 2% salt, so the condition with 2% salt is one in which NaCl has not been added to the medium. Microbial growth/survival was monitored through absorbance measurements in Bioscreen C, which measures bacterial growth over time by optical density (OD) measurements, for 7 days. Bioscreen C was programmed at a constant temperature of 30°C, with an hourly reading frequency and a wideband filter of 420–580 nm.

In addition, another set of experiment in Bioscreen was made at the same time in order to check if the real olive brine (extracted from sterilized jars with olives after 14 days in contact with the fruit and previously sterilized) was responsible for inhibiting growth. The real olive brine was characterized yielding the following levels: pH of 6.8, concentration of NaCl 6.28%, free acidity of 0.02, combined acidity of 0.05, and total sugars of 9.64% (sucrose 0.27%, glucose 6.26%, fructose 1.66%, and mannitol 1.45%). This real olive brine was modified in different conditions of pH levels (4, 5, 6, 7, 8, and 9) for the growth curve study.

The microtiter wells were filled with 340 μl of real olive and 10 μl of the Vibrio cocktail at a concentration of 10⁵ CFU/ml. Bioscreen C was programmed as described above.

At the second part of the experiment, the olives were inoculated with the pathogen and LAB. For this, four specific conditions have been proposed with their respective positive (samples inoculated with the target microorganism at time 0) and negative controls (non-inoculated samples):

The non-sterile brine was used in one of the conditions to know the possible effect of the accompanying olive microflora on Vibrio. Each condition was performed three times.