Formation of lactic acid bacteria–yeasts communities on the olive surface during Spanish-style Manzanilla fermentations

This work examines the formation of poly-microbial communities adhered to the surface of Manzanilla olive fruits processed according to the Spanish style. The experimental design consisted of four pilot fermenters inoculated with four Lactobacillus pentosus strains, plus another fermenter which was not inoculated and fermented spontaneously. Lactic acid bacteria and yeasts were analysed in depth on olive epidermis throughout fermentation by plate count, molecular techniques and scanning electron microscopy. Data show that in all cases high population levels (above 8 log10 CFU per olive) were reached for both groups of microorganisms at the second week of fermentation and that these counts never fell below 6 log10 CFU per olive during the 3 months that fermenters were monitored. In situ observation of olive epidermis slices revealed a strong aggregation and adhesion between bacteria and yeasts by the formation of a matrix which embedded the microorganisms. Geotrichum candidum, Pichia galeiformis and Candida sorbosa were the main yeast species isolated from these biofilms at the end of fermentation (confirmed by RFLP analysis of the 5.8S-ITS region), while molecular characterization of lactobacilli isolates by means of RAPD-PCR with primer OPL5 showed in many cases a high similarity in their banding profiles with the inoculated strains. Results obtained in this survey show the importance of studying the olive epidermis throughout fermentation, because ultimately, olives are ingested by consumers. ► Lactic acid bacteria and yeasts were analysed in depth on fermented olive epidermis. ► A strong aggregation and adhesion between bacteria and yeasts on olive epidermis was noticed. ► Microorganisms were embedded by an apparent exopolysaccharide matrix forming a complex biofilm. ► In the majority of cases, the inoculated Lactobacillus pentosus strains were imposed over the native LAB population.