Use of Lactiplantibacillus strains and yeasts for the production of fermented table olives and extra virgin olive oil

Table olives and olive oil are vegetable products, widespread among the Mediterranean countries, obtained, respectively, from microbial fermentation and extraction of olive tree fruits (Olea europea L.). According to the International Olive Oil Council, olives (green, black and turning colour) are mainly processed as alkali-treated olives (Spanish-style, treatment with 1.5-3.0% w/v NaOH and fermentation in brine with 10-11% w/v of salt for 3-7 months), natural olives (Greek-style, directly fermented in brine with 6-10% w/v of salt for 8-12 months), olives darkened by oxidation (Californian-style, fermented or not, with preservation in brine, darkening by oxidation in an alkaline medium and sterilization), olives dehydrated and/or shrivelled (exposed or not to a mild alkaline treatment, stored in brine or partially dehydrated in dry salt and/or by heating or other technological process) and specialties (olives prepared by using other processes). Microbial population of table olives mainly reflects that of olive fruits. Furthermore, industrial practices (e.g., equipment used, production environment) and processing methods (e.g., salt content, temperature) play an important role on the selection of microbiota. Fermentation can occur spontaneously, because of the development of the microbiota colonizing fresh olives, industrial environment, and processing tools and equipment, or for the addition of starter cultures (driven fermentation). Generally, olive fermentation is carried out by lactic acid bacteria (LAB; mainly Lpb. plantarum, and Lpb. pentosus) and/or yeasts (e.g., C. boidinii, P. membranifaciens, W. anomalus). LAB promote brine acidification, synthesis of antimicrobial compounds, degradation of oleuropein and formation of antioxidant compounds (e.g., hydroxytyrosol). Yeasts may impair (e.g., clouding of brines, production of metabolites related to off-flavours, off-odours, gas-pocket defects) or improve (e.g., production of metabolites that positively affect flavour and texture, improvement the LAB growth through the degradation of olives-related phenolic compounds) the quality of table olives. To date, the production of table olives is mainly based on spontaneous fermentations, that may have some disadvantages compared to driven process with starter cultures. Extra virgin olive oil is obtained from the fruits of the olive tree exclusively by mechanical or other physical methods under conditions that do not lead to alteration in the oil. The microbiological profile of olive oil partially reflects that of healthy and/or undamaged fruits. During the extraction process, the microbiota of olives carposphere migrates into olive oil, although microorganisms from oil mill environment and processing may also contribute to the composition of oil microbiota. During oil production and storage, microorganisms undergo selective pressures (mainly related to the presence of phenolic compounds and low sugar content) that may significantly affect the evolution of microbiota. Microorganisms are entrapped in micro-drops of vegetation water and their occurrence depends on capability to survive to oil-related conditions. Yeasts (e.g., C. oleophila, C. boidinii, N. molendinolei, N. wickerhamii, S. cerevisiae, Y. terventina) are the main microbial group able to survive over the storage period, while bacteria suffer more the toxic effect of phenolic compounds and rapidly decrease during the storage. Moulds may be found in low-quality oils extracted from damaged fruit. Based on their metabolic activities, yeasts may positively (e.g., β-glucosidase and esterase enzymes) or negatively (e.g., phenoloxidases, peroxidases, lipases, pectinases enzymes) affect the physicochemical and sensory quality of the olive oil. Yeasts activities also affect the concentration and content of volatile organic compounds (VOCs) that contribute to the sensory profile of EVOO (Chapter 1). In this contest, the PhD thesis is focused on the identification, characterization and selection of suitable Lactiplantibacillus strains to be used as starter cultures during table olive fermentation. A further aim of this study was to improve knowledge on the biodiversity and viability of yeasts in olive oil samples, as well as reach information about their effect on olive oil quality. A large collection of Lactiplantibacillus strains was screened for useful technological properties (i.e., xylose fermentation, exopolysaccharides production, antimicrobial activity, tolerance to phenolic compounds, oleuropein degradation and hydroxytyrosol formation, survival to olive mill wastewater and simulated gastro-intestinal tract, capability to grow at different combinations of NaCl and pH values, radical scavenging activities and biofilm formation) relevant for the production of fermented table olives. The screening step revealed high diversity among Lactiplantibacillus strains. Most of strains were able to ferment xylose, while only few strains produced EPS and had inhibitory activity against Yarrowia lipolytica. Resistance to phenolic compounds (gallic, protocatechuic, hydroxybenzoic and syringic acids), as well as ability to released hydroxytyrosol from oleuropein were strain-specific. Olive mill wastewater (OMW) impaired strains survival, while combinations of NaCl ≤ 6% and pH ≥ 4.0 were well tolerated. Some selected strains were also able to produce biofilm, scavenge toxic radicals and survive to simulated GIT and simulated brine, suggesting their possible used as starter for production of fermented table olives (Chapter 2). The genome of four Lpb. pentosus strains (O11, O12, O17 and O19) with interesting technological features, was sequenced and analysed to verify the presence of genes involved in degradation and metabolism of phenolic compounds. The genomes harbour most of the abovementioned genes and lack only the sequences encoding for carboxylesterase and gallate decarboxylase subunits B and D. Lpb. pentosus O12 also lacks the sequence for the subunits A of tannase gene (Chapter 3). Lpb. pentosus O17 was selected and used as starter culture for the production of natural (Itrana table olives) and alkali-treated (Manzanilla table olives and compared with the starter culture Lpb. pentosus LPG1) table olives. Spontaneous processes were used as control for both Itrana and Manzanilla table olives. For Itrana process, the evolution of pH, titratable acidity, NaCl and phenolic compounds contents (TPC), as well as of different microbial groups (total bacteria, LAB, yeasts, moulds, Enterobacteriaceae, halophilic LAB, yeasts and others bacteria) was monitored in both brines and olives, up to 6 months of fermentation. A sensory evaluation as well as a texture profile analysis and volatile organic compounds detection (E-nose measurement) were performed for final Itrana olives. The presence of Lpb. pentosus O17 boosted the decrease of pH, increased the titratable acidity and reduced the TPC compared to spontaneous fermentation. Addition of high salt concentrations impaired the viability of Lpb. pentosus O17, and yeasts population (tolerant to osmotic stress) became dominant and driven the fermentation process until the end of fermentation. During Manzanilla process, pH, titratable and combined acidity and salt concentration was measured in brines, while colour, firmness and moisture was assessed for olive fruits. The evolution of LAB, yeasts and enterobacteria was measured for both brine and olives. The viability of starter cultures (both Lpb. pentosus O17 and LPG1) was not impaired in alkali-treated fermentation and LAB was dominant until the end of the processes (35 days; Chapter 4). Regarding olive oil, 17 yeast strains belonging to Candida boidinii, Lachancea fermentati, Nakazawaea molendinolei, Nakazawaea wickerhamii and Schwanniomyces polymorphus species were collected during olive oil production, identified and tested for the ability to ferment sugars, to grow at low temperatures, for the occurrence of different enzymatic activities, for the tolerance and degradation of phenolic compounds, radical scavenging activities, biofilm formation, survival to simulated gastro-intestinal (GIT) tract. The characterized yeasts were also inoculated in extra virgin olive oils (EVOO; from Leccino and Coratina cultivar) to evaluate their survival and their effect on EVOO quality (changes in analytical indices) during 6-months of storage. High levels of survival were observed for almost strains (except S. polymorphus), in both Leccino and Coratina samples. Yeasts limited the acidity rise in olive oils, but overtime they contributed to increase the parameters related to oxidative phenomena (i.e. peroxides, K232, K270), resulting in a declassification of EVOOs. The total phenolic content was correlated to the presence of yeasts and, at the end of storage period (6 months) inoculated samples had significantly lower concentrations compared to the control oils (Chapter 5). In conclusion (Chapter 6), the achievement of research activities foreseen in this PhD thesis allowed to obtain the following results: - scientific results: 1) information on the diversity within the strains and species of the genus Lactiplantibacillus; 2) knowledge on the survival of Lactiplantibacillus species to phenolic compounds; 3) information on the occurrence of genes involved in phenolic compound metabolism in Lpb. pentosus species; 4) availability of 4 Lpb. pentosus genomes useful to provide further physiological and genomes information that, to date, are significant lower compared to the closely species Lpb. plantarum. - technological and applicative results: 1) availability of well-characterized Lpb. pentosus O17 to be used as starter culture for the production of fermented table olives; 2) information on the effect of LAB and yeasts on the quality of olive derived products; 3) the provided further information on the evolution of olive oil microbiota, useful to better monitoring and fine-tune the production process of this product. Future prospects On the basis of the obtained results, further studies on the occurrence and genetic polymorphism of genes involved in metabolism and tolerance of phenolic compounds, as well as of other interesting genes (e.g., genes involved in acid and osmotic stresses) for the production of fermented table olives, should be carried out to better highlight the functionalities of selected Lpb. pentosus strains. Additionally, further metataxonomic and metagenomic studies should be carried out to investigate the diversity and the evolution of microbiota during the production of fermented table olives, in order to optimised targeted fermentation that could be provided more benefits compered to spontaneous and uncontrolled fermentations. Further studies are also needed to improve knowledge on the factors that affecting microbial survival in olive oil, to better monitoring and fine-tune the production processes of this product.