Table Olive Fermentation Research Articles

Probiotic potential of indigenous yeasts isolated during the fermentation of table olives from Northeast of Portugal

Abstract The aim of this study was to evaluate the probiotic potential of 16 yeast strains previously isolated during the natural fermentation of table olives Negrinha de Freixo cultivar, in relation to enzymatic activities; ability to grow at 37 °C; antimicrobial activity; autoaggregation capacity; antioxidant activity and survival in gastrointestinal tract conditions. The highest antioxidant activity was observed for Saccharomyces cerevisiae, similar to the reference strain S. boulardii. Candida norvegica 7A and Galactomyces reessii 34A showed antifungal ability to the pathogenic microorganism Cryptococcus neoformans. Regarding the autoaggregation capacity, S. cerevisiae 15A, C. tropicalis 1A and C. norvegica 7A showed > 80% after 24 h. Pichia guilliermondii 25A and C. norvegica 7A were the most resistant to the simulated digestive conditions, similar to the reference strain (S. boulardii). Thus, these results suggest that some yeast strains involved in the fermentation of table olives have probiotic potential. Industrial relevance In this study, we highlight the probiotic potential of yeast microbiota usually found in green table olives. These yeast strains could be used as culture starters for the development of new functional products.

Evolution of microbiota during spontaneous and inoculated Tonda di Cagliari table olives fermentation and impact on sensory characteristics

Starters are widely used for driving food fermentations faster and more safely. Recently, the use of natural autochthonous microbiota is being reappraised for obtaining products with peculiar and unique characteristics. The aim of this study was to investigate, by DGGE and REP-PCR(GTG)5, the microbial dynamics and intra-species biodiversity of Tonda di Cagliari table olives inoculated with a mix of autochthonous Lactobacillus pentosus strains (SIE) or a single Lactobacillus plantarum strain (SSL), taking spontaneous fermentation (NF) as control. Furthermore, the sensory quality of the three fermentations trials was evaluated by Descriptive Analysis. Among 316 isolates, 289 were identified as Lactobacillus pentosus, and only 31, coming exclusively from batches SSL, as Lactobacillus plantarum. The predominance of L. pentosus can be explained by their better adaptation to the olive fermentation environment with respect to L. plantarum selected strain inoculated in SSL fermentations. Using the culture independent approaches, differences in the fermentation processes were shown only during the first months of fermentation, and several Gram negative species, not frequently isolated in these ecosystems (Thalassomonas agarivorans, non-fermentative bacteria, and the cyanobacteria Chroococcidiopsis), were identified. Autochthonous starter SIE, compared to SSL, produced olives more similar, in sensory attributes, to the naturally fermented ones.

Table Olive Fermentation Using Starter Cultures with Multifunctional Potential.

Table olives are one of the most popular plant-derived fermented products. Their enhanced nutritional value due to the presence of phenolic compounds and monounsaturated fatty acids makes olives an important food commodity of the Mediterranean diet. However, despite its economic significance, table olive fermentation is mainly craft-based and empirically driven by the autochthonous microbiota of the olives depending on various intrinsic and extrinsic factors, leading to a spontaneous process and a final product of variable quality. The use of microorganisms previously isolated from olive fermentations and studied for their probiotic potential and technological characteristics as starter cultures may contribute to the reduction of spoilage risk resulting in a controlled fermentation process. This review focuses on the importance of the development and implementation of multifunctional starter cultures related to olives with desirable probiotic and technological characteristics for possible application on table olive fermentation with the main purpose being the production of a health promoting and sensory improved functional food.

Assessing the Challenges in the Application of Potential Probiotic Lactic Acid Bacteria in the Large-Scale Fermentation of Spanish-Style Table Olives.

This work studies the inoculation conditions for allowing the survival/predominance of a potential probiotic strain (Lactobacillus pentosus TOMC-LAB2) when used as a starter culture in large-scale fermentations of green Spanish-style olives. The study was performed in two successive seasons (2011/2012 and 2012/2013), using about 150 tons of olives. Inoculation immediately after brining (to prevent wild initial microbiota growth) followed by re-inoculation 24 h later (to improve competitiveness) was essential for inoculum predominance. Processing early in the season (September) showed a favorable effect on fermentation and strain predominance on olives (particularly when using acidified brines containing 25 L HCl/vessel) but caused the disappearance of the target strain from both brines and olives during the storage phase. On the contrary, processing in October slightly reduced the target strain predominance on olives (70–90%) but allowed longer survival. The type of inoculum used (laboratory vs. industry pre-adapted) never had significant effects. Thus, this investigation discloses key issues for the survival and predominance of starter cultures in large-scale industrial fermentations of green Spanish-style olives. Results can be of interest for producing probiotic table olives and open new research challenges on the causes of inoculum vanishing during the storage phase.

Towards Controlled Fermentation of Table Olives: LAB Starter Driven Process in an Automatic Pilot Processing Plant

The results of an experimental trial, carried out using a specifically prepared microbial starter, in an automated pilot plant, are presented. The microbial starter was composed by Lb. pentosus strains. The automatized process was compared with natural fermentation in plastic vats, as control. Relevant process parameters, chemico-physical determinations, instrumental texture, and microbiological analyses were carried out to monitor the fermentation process. Driving the process in controlled conditions resulted in a more rapid acidification, pH reaching lower values than the control. In controlled conditions, inoculated lactobacilli carried out a fast colonization supplanting more rapidly spoilage microflora. In started samples, yeasts number increased until reaching 5 log at 60 days, keeping this value constant until the end of the trials, while, in control batches, their growth was more rapid, reaching 5 log from 15 to 30 days, but their number decreased to 3 log at 90 days. The two processes produced olives with comparable texture profile. However, olives coming from the controlled process resulted debittered in 3 months (descriptive sensory analyses and oleuropein quantification in HPLC), while naturally fermented olives resulted not yet debittered at the end of the trial (180 days). Slight differences were found in few other sensory descriptors. Our results showed that, along with the use of effective autochthonous microbial starters, the innovation of processing plants is the next step towards the shortening of the processing time and the safety assurance of the product, while retaining the sensory quality of naturally fermented olives, using an environmentally friendly technology.

Microbiota and metabolome during controlled and spontaneous fermentation of Nocellara Etnea table olives

This study is aimed to investigate bacterial community and its dynamics during the fermentation of Nocellara Etnea table olives and to study its effect on metabolome formation. Six different combination of bacterial cultures (BC1-BC6) were used as starters for table olive fermentation and one additional process, conducted without addition of any starters, was used as control (C). The processes were conducted in triplicate and, overall, 21 vessels were performed at industrial scale. The fermentation was monitored for 120 days through culture-dependent and –independent approaches. Microbial counts of the main microbial groups revealed slight differences among brine samples, with the exception of LAB counts and Enterobacteriaceae, which were higher and lower, respectively, in most of the inoculated samples than the control ones. In addition, results demonstrated that the use of bacterial cultures (except the BC1), singly or in different combinations, clearly influenced the fermentation process reducing the final pH value below 4.50. When microbiota was investigated through sequencing analysis, data revealed the presence of halophilic bacteria and, among lactobacilli, the dominance of Lactobacillus plantarum group at the initial stage of fermentation, in all brine samples, except in the BC5 in which dominated Lactobacillus casei group. At 60 and 120 days of fermentation, an overturned bacterial ecology and an increase of biodiversity was observed in all samples, with the occurrence of Lactobacillus paracollinoides, Lactobacillus acidipiscis and Pediococcus parvulus. Correlation between bacterial OTU and volatile organic compounds (VOCs) revealed that, aldehydes and alcohol compounds exhibited a positive correlation with Proteobacteria, while several esters with LAB and Hafnia. In particular, esters, associated with fruity and floral notes, were positively correlated to L. paracollinoides, L. acidipiscis, and P. parvulus species. Although the VOCs amounts were sample-specific, overall aldehydes were mostly produced at the beginning of the fermentation, while acids, alcohols and esters at the end of the process.

Investigating the Effect of Different Treatments with Lactic Acid Bacteria on the Fate of Listeria monocytogenes and Staphylococcus aureus Infection in Galleria mellonella Larvae.

The use of Galleria mellonella as a model host to elucidate microbial pathogenesis and search for novel drugs and therapies has been well appreciated over the past years. However, the effect of microorganisms with functional appeal in the specific host remains scarce. The present study investigates the effect of treatment with selected lactic acid bacteria (LAB) with probiotic potential, as potential protective agents by using live or heat-killed cells at 6 and 24 h prior to infection with Listeria monocytogenes and Staphylococcus aureus or as potential therapeutic agents by using cell-free supernatants (CFS) after infection with the same pathogens. The employed LAB strains were Lactobacillus pentosus B281 and Lactobacillus plantarum B282 (isolated from table olive fermentations) along with Lactobacillus rhamnosus GG (inhabitant of human intestinal tract). Kaplan-Meier survival curves were plotted while the pathogen’s persistence in the larval hemolymph was determined by microbiological analysis. It was observed that the time (6 or 24 h) and type (live or heat-killed cells) of challenge period with LAB prior to infection greatly affected the survival of infected larvae. The highest decrease of L. monocytogenes population in the hemolymph was observed in groups challenged for 6 h with heat-killed cells by an average of 1.8 log units compared to non challenged larvae for strains B281 (p 0.0322), B282 (p 0.0325), and LGG (p 0.0356). In the case of S. aureus infection, the population of the pathogen decreased in the hemolymph by 1 log units at 8 h post infection in the groups challenged for 6 h with heat-killed cells of strains B281 (p 0.0161) and B282 (p 0.0096) and by 1.8 log units in groups challenged with heat-killed cells of LGG strain (p 0.0175). Further use of CFS of each LAB strain did not result in any significant prolonged survival but interestingly it resulted in pronounced decrease of L. monocytogenes in the hemolymph at 24 h and 48 h after infection by more than 1 log unit (p < 0.05) depending on the strain. The results of the present work support the broader use of G. mellonella larvae as a low cost in vivo tool for screening for probiotic properties.

Assessment of table olive fermentation by functional data analysis

For the first time, functional data analysis (FDA) was used to assess the effects of different treatments on Protection Denomination of Origin Aloreña de Málaga table olive fermentations, focusing on the evolution of yeast population. The analysis of fermentation by a conventional approach led to scarce information. However, the transformation of microbial (and also physicochemical) data into smooth curves allowed the application of a new battery of statistical tools for the analysis of fermentations (functional pointwise estimation of the averages and standard deviations, maximum, minimum, first and second derivatives, functional regression, and functional F and t-tests). FDA showed that all the treatments assayed led to similar trends in yeast population while changes in pH and titratable acidity profiles led to several significant differences. Therefore, FDA represents a promising and valuable tool for studying table olive fermentations and for food microbiology in general.

RT-PCR-DGGE Analysis to Elucidate the Dominant Bacterial Species of Industrial Spanish-Style Green Table Olive Fermentations.

This paper describes the dominant bacterial species metabolically active through the industrial production of Spanish-style Manzanilla and Gordal olives. For this purpose, samples (brines and fruits) obtained at 0, 15, and 90 fermentation days were analyzed by a culture-independent approach to determine viable cells by reverse transcription of RNA and further PCR-DGGE analysis, detecting at least 7 different species. Vibrio vulnificus, Lactobacillus plantarum group, and Lactobacillus parafarraginis were present in samples from both cultivars; Lactobacillus sanfranciscensis and Halolactobacillus halophilus were detected only in Gordal samples, while Staphylococcus sp. was exclusively found at the onset of Manzanilla fermentations. Physicochemical data showed a typical fermentation profile while scanning electron microscopy confirmed the in situ biofilm formation on the olive epidermis. Different Bacillus, Staphylococcus, and Enterococcus species, not detected during the fermentation process, were also found in the solid marine salt used by the industry for preparation of brines. Elucidation of these non-lactic acid bacteria species role during fermentation is then an appealingly challenge, particularly regarding safety issues.

Assessment of the bacterial community in directly brined Aloreña de Málaga table olive fermentations by metagenetic analysis

This study uses an “omics” approach to evaluate the bacterial biodiversity changes during fermentation process of natural green cracked Aloreña de Málaga table olives, from raw material to fermented fruit. For this purpose, two industries separated by almost 20km in Guadalhorce Valley (Málaga, Spain) were analysed for obtaining both brines and fruit samples at different moments of fermentation (0, 7, 30 and 120days). Physicochemical and microbial counts during fermentation showed the typical evolution of this type of processes, apparently dominated by yeasts. However, high-throughput barcoded pyrosequencing analysis of V2–V3 hypervariable region of the bacterial 16S rRNA gene showed at 97% identity the presence of 131 bacterial genera included in 357 operational taxonomic units, not detected by the conventional approach. The bacterial biodiversity was clearly higher in the olives at the moment of reception in the industry and during the first days of fermentation, while decreased considerably as elapse the fermentation process. The presence of Enterobacteriaceae and Lactobacillaceae species was scarce during the four months of study. On the contrary, the most important genus at the end of fermentation was Celerinatantimonas in both brine (95.3% of frequency) and fruit (89.4%) samples, while the presence of well-known spoilage microorganisms (Pseudomonas and Propionibacterium) and halophilic bacteria (Modestobacter, Rhodovibrio, Salinibacter) was also common during the course of fermentation. Among the most important bacterial pathogens related to food, only Staphylococcus genus was found at low frequencies (<0.02% of total sequences). Results show the need of this type of studies to enhance our knowledge of the microbiology of table olive fermentations. It is also necessary to determine the role played by these species not previously detected in table olives on the quality and safety of this fermented vegetable.

Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations.

The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of "sticky" material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing.

In Lactobacillus pentosus, the olive brine adaptation genes are required for biofilm formation

Lactobacillus pentosus is one of the few lactic acid bacteria (LAB) species capable of surviving in olive brine, and thus desirable during table olive fermentation. We have recently generated mutants of the efficient strain L. pentosus C11 by transposon mutagenesis and identified five mutants unable to survive and adapt to olive brine conditions. Since biofilm formation represents one of the main bacterial strategy to survive in stressful environments, in this study, the capacity of adhesion and formation of biofilm on olive skin was investigated for this strain and five derivative mutants which are interrupted in metabolic genes (enoA1 and gpi), and in genes of unknown function (“oba” genes). Confocal microscopy together with bacteria count revealed that the sessile state represented the prevailing L. pentosus C11 life-style during table olive fermentation. The characterization of cell surface properties showed that mutants present less hydrophobic and basic properties than the wild type (WT). In fact, their ability to adhere to both abiotic (polystyrene plates) and biotic (olive skin) surfaces was lower than that of the WT. Confocal microscopy revealed that mutants adhered sparsely to the olive skin instead of building a thin, multilayer biofilm. Moreover, RT-qPCR showed that the three genes enoA1, gpi and obaC were upregulated in the olive biofilm compared to the planktonic state. Thus enoA1, gpi and “oba” genes are necessary in L. pentosus to form an organized biofilm on the olive skin.

Evaluation of Two Lactic Acid Bacteria Starter Cultures for the Fermentation of Natural Black Table Olives (Olea europaea L cv Kalamon)

The production of Greek-style natural black table olives remains an empirical process relying on spontaneous fermentation despite its economic significance. For this reason producers often resort to increased NaCl concentration of the brine to secure quality of the product. In this study we employ two lactic acid bacteria Leuconostoc mesenteroides subsp. mesenteroides Lm139 and Lactobacillus pentosus DSM 16366 as starters in separate laboratory low salinity fermentations of "Kalamon" cultivar olives, processed according to the Greek-style method. L. mesenteroides subsp. mesenteroides Lm139 was previously isolated from Kalamon olives laboratory spontaneous fermentations, while L. pentosus DSM 16366 was isolated from fermenting green olives prepared according to the Spanish-style method. Spontaneous olives fermentation was also performed as a control. Microbiological and physicochemical analyses of the brines revealed that the use of the starters had a significant effect on the olives fermentation, leading to a faster acidification due to the more efficient consumption of soluble sugars in the brines. The final pH value reached by each starter culture used indicates a successful lactic fermentation. The production of lactic acid by the starters and the concomitant drop of the pH value proved to inhibit enterobacteria in a shorter period of time compared to the spontaneous fermentation. Concluding, the use of either of the two lactic acid bacteria as starters in Greek-style Kalamon olives fermentation could lead to a more controllable fermentation at lower salinities. The resulting product could be of higher quality with extended shelf-life while being at the same time safer for the consumer.

Diversity and enumeration of halophilic and alkaliphilic bacteria in Spanish-style green table-olive fermentations

The presence and enumeration of halophilic and alkaliphilic bacteria in Spanish-style table-olive fermentations was studied. Twenty 10-tonne fermenters at two large manufacturing companies in Spain, previously studied through both culture dependent and independent (PCR-DGGE) methodologies, were selected. Virtually all this microbiota was isolated during the initial fermentation stage. A total of 203 isolates were obtained and identified based on 16S rRNA gene sequences. They belonged to 13 bacterial species, included in 11 genera. It was noticeable the abundance of halophilic and alkaliphilic lactic acid bacteria (HALAB). These HALAB belonged to the three genera of this group: Alkalibacterium, Marinilactibacillus and Halolactibacillus. Ten bacterial species were isolated for the first time from table olive fermentations, including the genera Amphibacillus, Natronobacillus, Catenococcus and Streptohalobacillus. The isolates were genotyped through RAPD and clustered in a dendrogram where 65 distinct strains were identified. Biodiversity indexes found statistically significant differences between both patios regarding genotype richness, diversity and dominance. However, Jaccard similarity index suggested that the halophilic/alkaliphilic microbiota in both patios was more similar than the overall microbiota at the initial fermentation stage. Thus, up to 7 genotypes of 6 different species were shared, suggesting adaptation of some strains to this fermentation stage. Morisita-Horn similarity index indicated a high level of codominance of the same species in both patios. Halophilic and alkaliphilic bacteria, especially HALAB, appeared to be part of the characteristic microbiota at the initial stage of this table-olive fermentation, and they could contribute to the conditioning of the fermenting brines in readiness for growth of common lactic acid bacteria.

Evaluation of Toma Piemontese PDO cheese as a carrier of putative probiotics from table olive fermentations

Two strains of Lactobacillus plantarum (Lp S11T3E and Lp S2T10D) and one of Lactobacillus pentosus (Lps S3T60C), originally isolated from table olive fermentations, and previously characterized for their probiotic properties, have been added as adjunct cultures to Toma Piemontese Protected Denomination of Origin (PDO) cheese, in order to develop a functional dairy product. The cheeses were sampled throughout their manufacturing process and during the two months of ripening. Moreover, they were also subjected to in vitro human digestion, followed by microbiological analysis. Monitoring of the inoculated strains was performed through molecular identification and biotyping. The organic acid and sugar contents of the cheeses were determined at each sampling point, while the organoleptic features were assessed by sensory evaluation. All isolated lactobacilli, during ripening and after digestion of the cheeses, were recognized as putative probiotics, and demonstrated to be well adapted to the food matrix investigated. The organic acid composition of the cheeses with the adjunct culture differed from the control, but this difference did not negatively affect the organoleptic profiles of the final product. Therefore, all the strains tested were to be usable as adjunct cultures in Toma Piemontese PDO production to develop a new functional food.

Effect of the mechanical harvest of drupes on the quality characteristics of green fermented table olives.

Because of damage caused by mechanical harvesting, the drupes for table olive production are traditionally hand harvested. Until now, no data have been available on the microbiological and chemical features of mechanically harvested drupes during fermentation. Drupes mechanically harvested and inoculated with Lactobacillus pentosus OM13 were characterized by the lowest concentrations of potential spoilage microorganisms. On the other hand, drupes mechanically harvested and subjected to spontaneous fermentation showed the highest concentration of Enterobacteriaceae and pseudomonads during transformation. The lowest decrease of pH (4.20) was registered for the trials inoculated with the starter culture. Differences in terms of volatile organic compounds were estimated among trials. Multivariate analysis showed that the olives processed from the drupes mechanically harvested and inoculated with starter were closely related to control production (drupes manually harvested) in terms of microbiological and pH values. Sensory analysis evidenced negative evaluations only for the uninoculated trials. Drupes mechanically harvested and subjected to a driven fermentation with Lactobacillus pentosus OM13 determined the production of table olives with appreciable organoleptic features. Thus mechanical harvesting performed using a trunk shaker equipped with an inverse umbrella and the addition of starter lactic acid bacteria represents a valuable alternative to manual harvesting for table olive production at the industrial level.

Fermentation of table olives by oleuropeinolytic starter culture in reduced salt brines and inactivation of Escherichia coli O157:H7 and Listeria monocytogenes

The effect of an autochthonous starter culture developed by oleuropeinolytic strains belonging to the Lactobacillus plantarum group on the physicochemical and microbiological characteristics and the biophenol content of table olives fermented under reduced salt conditions was studied. Black (cv. Kalamata) and green (cv. Chalkidikis) olives were fermented in two different kinds of brine (Brine A containing 2.3% NaCl, 32.3mM Ca-acetate and 33.9mM Ca-lactate and Brine B containing 4% NaCl, pH5.0 in both brines). The sensory attributes of olives fermented by oleuropeinolytic starter culture assessed by a trained panel did not differ significantly compared with industrial processing. It is possible to carry out significant changes in table olive processing applying a completely microbiological procedure using oleuropeinolytic strains of the L. plantarum group as both the debittering and the fermentation agent in order to achieve improved sensorial and nutritional characteristics of the final product. Table olives processed by the suggested methodology may constitute a good source of biophenols in the diet, especially hydroxytyrosol and tyrosol. The inactivation potential of Escherichia coli O157 EDL-932 and Listeria monocytogenes Scott A in olives fermented by oleuropeinolytic starter culture was evaluated. The population of each pathogen in olive homogenates of both cultivars is inactivated by more than 6logCFU/ml in less than 24h. When whole fermented olives were submerged in peptone/saline (containing 6.7logCFU/ml of the relevant bacterial pathogen) for 30min followed by rinsing in distilled water, the population of viable foodborne pathogens dropped more than 4 logs in olive pulp. During subsequent storage at 22 or 4°C the population of L. monocytogenes Scott A was further eliminated under the detection limit in both olive cultivars whereas the population of E. coli O157 EDL-932 could be detected in olives stored in peptone/saline at 22°C for 7days. The inhibitory effect of olives fermented by oleuropeinolytic starter culture in reduced salt brines on pathogens is due to the antimicrobial activity of the phenolic compounds and the antagonistic action of the associated microflora.

Acceleration of ripening and improvement of quality of olive pickles by encapsulated cells in nano/micro-tubular cellulose

An industrial process for black table olives fermentation was followed in order to study the effect of immobilized halophile culture and the initial halophile culture concentration in combination with temperature for a period of 30 days. For comparison, three types of batch fermentation were investigated: (a) fermentation with the indigenous microbiota (control), (b) inoculated fermentation with isolated mixed halophile culture (MHC) and (c) inoculated fermentation with isolated MHC on tubular cellulose (TC). The effects of (i) temperature (5 and 20 °C) and (ii) initial biomass concentration (0.15, 0.5, 1 and 1.5 g/300 ml brine) on pH, brine and total volatiles were studied. Immobilized MHC cells on TC increased the fermentation rate as compared with free MHC cells. When immobilized MHC was used in fermentation, pH values remained close to 4.0 for all initial culture concentrations and lower than that for natural fermentation for 30 days at 5 °C. Especially for 1 g of biomass/300 ml of brine initial culture, the pH remained lower than 3.4 during the 30 days of fermentation at 5 °C. At the case of immobilized halophile cells at 5 °C, propionic acid was produced (max. 26.74 ± 1.21 g/L) which acted as natural preservative, while there was increase in total volatiles.

PCR-DGGE assessment of the bacterial diversity in Spanish-style green table-olive fermentations

The bacterial ecology associated to Spanish-style green olive fermentations has been studied, attending to its dynamics along the time and its distribution, by a culture-independent approach based on PCR-DGGE. Forty-three 10-tonne fermenters were selected from the fermentation yards (patios) of two large table-olive manufacturing companies in southern Spain. The fermenting brines of 20 of these fermenters were previously analyzed through culture-dependent methods, allowing comparisons of both methodologies. A statistical analysis of DGGE banding profiles obtained using bacteria universal primers demonstrated significant evidences of discrimination of bacterial communities by location (patio) and fermentation stage. Specific microbial “fingerprints” could be established for these variables. At least 17 bacterial species were detected, most of them previously isolated from the same fermenters. Most of these species belonged to the lactic acid bacteria (LAB) group. Dominance of species within the Lactobacillus plantarum group was confirmed. Marinilactibacillus sp. and Propionibacterium olivae, which were not isolated in the previous culture-dependent study, were detected. Alkalibacterium sp. and Halolactibacillus sp. were detected for the first time in table olive fermentations. Using Lactobacillus-group specific primers, significant clustering within the DGGE banding profiles was observed, allowing discrimination regarding the actual fermentation stage. These results corroborated the previous culture-dependent study, and added the detection of Alkalibacterium sp. and Pediococcus acidilactici. The species Alkalibacterium sp., Marinilactibacillus sp. and Halolactibacillus sp. are characterized by their ability to carry out lactic acid fermentation under alkaline conditions and thus ascribed within the halophilic and alkaliphilic lactic acid bacteria (HALAB). Their ubiquitous presence suggests that they could play an important role in Spanish-style olive fermentations, especially at the initial fermentation stage. Thus, they could contribute to brine conditioning before L. plantarum group-driven lactic acid fermentation takes place.

An innovative method to produce green table olives based on “pied de cuve” technology

The technology of “pied de cuve” (PdC) is applied in food process only to produce wines with an enriched community of pro-technological yeasts. PdC promotes the growth of the desirable microbial strains in a small volume of grape must acting as a starter inoculums for higher volumes. The aim of the present work was to investigate the use of partially fermented brines, a technology known as PdC, developed with lactic acid bacteria (LAB) on the microbiological, chemical and sensory characteristics of green fermented table olives during two consecutive campaigns. The experimental plan included two trials based on different PdCs: trial A, PdC obtained with Lactobacillus pentosus OM13; trial B, PdC obtained through a spontaneous fermentation. Two control additional trials without PdC were included for comparison: trial C, spontaneous fermentation; trial D, direct inoculation of L. pentosus OM13. The use of PdCs favoured the rapid increase of LAB concentrations in both trials A and B. These trials showed levels of LAB higher than trial C and almost superimposable to that of trial D. Trial B was characterized by a certain diversity of L. pentosus strains and some of them dominated the manufacturing process. These results indicated PdC as a valuable method to favour the growth of autochthonous L. pentosus strains. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) visibly discriminated olive processes fermented with the two experimental PdCs. Interestingly, on the basis of microbial and pH variables, both approaches showed that the olives produced with PdC technology are closely related to those of trial D, with the advantages of reducing the amount of starter to inoculate (trial A) and a higher LAB biodiversity (trial B). Volatile organic compound (VOC) composition and sensory analysis showed trials A and B different from the trials with no PdC added, in both years. Furthermore, the trial B showed the highest scores of green olive aroma and taste complexity. Spoilage microorganisms were estimated at very low levels in all trials. Undesired off-odours and off-flavours were not revealed at the end of the process.