Mono and Dual Species Biofilm Formation Between Lactobacillus Pentosus and Pichia Membranifaciens on the Surface of Black Olives under Different Sterile Brine Conditions

The purpose of this work was to investigate the in situ predisposition of Lactobacillus pentosus B281 grown in both monoculture and co-culture with the yeast Pichia membranifaciens M3A to adhere on the surface of black olives and develop single and mixed microbial communities under different sterile brine conditions. Black oxidized olives were submerged in brine solution and inoculated with an initial load of 5.0 log CFU/ml L. pentosus or with a combined culture of 5.0 log CFU/ml L. pentosus and P. membranifaciens. Two initial salt concentrations in the brines were investigated, namely 6 % and 10 %, corresponding to low and high salt brine, respectively. Brines were supplemented with (1) 0.2 % (v/v) lactic acid, (2) 0.5 % (w/v) glucose, and (3) both 0.2 % (v/v) lactic acid and 0.5 % (w/v) glucose. A brining treatment with no supplementation of glucose and lactic acid was also studied as a control treatment. Each brining condition was studied in duplicate at 20 °C for a period of 30 days. The population dynamics of the inoculated strains on the surface of olives were determined by plate count, whereas olive samples were observed at the end of storage under scanning electron microscopy (SEM). Results showed that, in the case of low salt brines, high population levels between 6.5 and 7.0 log CFU/g were reached on olive drupes for L. pentosus in both single and mixed culture inoculation regardless of brining treatment. However, in high salt brines no cells of the microorganism could be recovered from the control and glucose supplemented brines in the case of single culture inoculation as well as in acidified brines with/without glucose in mixed inoculations. The presence of biofilm on the surface of olives was also confirmed by SEM. Aggregates of the lactic acid bacteria and the yeast could be observed located mostly in the stomatal apertures and on the epidermis.