Green Olive Fermentation Research Articles

The use of multifunctional yeast-lactobacilli starter cultures improves fermentation performance of Spanish-style green table olives

In this work, Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, and Wickerhanomyces anomalous Y12, all of them previously isolated from fermented table olive biofilms, were used (alone or in combination) as multifunctional starters for Manzanilla Spanish-style green table olive fermentations. Their performances were evaluated through the changes in the key physico-chemical and microbiological parameters, correlation between AI-2 production and biofilm formation, inoculum imposition, metataxonomic analysis and sensory characteristics of the finished products. Inoculation only with lactic acid bacteria (LAB) strains led to higher titratable acidities and lower pH values than the spontaneous fermentation (non-inoculated control), mainly during the first steps of processing. However, the sequential inoculation of the yeast and then the combination of the 3 LAB strains showed the most favourable evolution. LPG1 strain and, particularly Lp13, were excellent biofilms former and showed the major imposition on the fruit epidermis, as corroborated by rep-PCR analysis. Production of AI-2 was lower in the treatment inoculated exclusively with yeast Y12 but had the highest presence in the sequential yeast-LAB inoculum, with its maximum concentration and maximum LAB population on fruits (19th days) strongly related. Metataxonomic analysis of the biofilms at the end of the fermentation revealed, in addition to Lactobacillus, high proportions of sequences from genera Marinilactobacillus, Alkalibacterium, Halolactobacillus, and low levels of Halomonas and Aerococcus. Compositional data analysis of the omics data revealed that Lpl15 was scarcely efficient for controlling the spontaneous microbiota since its treatment presented the highest proportions of Aerococcus genus. Finally, the sensory analysis showed similar characteristics for the treatment inoculated with LPG1 and the spontaneous process, with olives inoculated with the yeast (alone or in combination with Lactobacillus strains) showing attractive scores. Then, inoculation of Spanish-style table olive fermentations with a sequential yeast and LAB combination could be an advisable practice.

Lactic Acid Bacteria and Yeast Inocula Modulate the Volatile Profile of Spanish-Style Green Table Olive Fermentations

In this work, Manzanilla Spanish-style green table olive fermentations were inoculated with Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, the yeast Wickerhanomyces anomalus Y12 and a mixed culture of all them. After fermentation (65 days), their volatile profiles in brines were determined by gas chromatography-mass spectrometry analysis. A total of 131 volatile compounds were found, but only 71 showed statistical differences between at least, two fermentation processes. The major chemical groups were alcohols (32), ketones (14), aldehydes (nine), and volatile phenols (nine). Results showed that inoculation with Lactobacillus strains, especially L. pentosus Lp13, reduced the formation of volatile compounds. On the contrary, inoculation with W. anomalus Y12 increased their concentrations with respect to the spontaneous process, mainly of 1-butanol, 2-phenylethyl acetate, ethanol, and 2-methyl-1-butanol. Furthermore, biplot and biclustering analyses segregated fermentations inoculated with Lp13 and Y12 from the rest of the processes. The use of sequential lactic acid bacteria and yeasts inocula, or their mixture, in Spanish-style green table olive fermentation could be advisable practice for producing differentiated and high-quality products with improved aromatic profile.

Multiple Genome Sequences of Lactobacillus pentosus Strains Isolated from Biofilms on the Skin of Fermented Green Table Olives.

The draft genome sequences of five Lactobacillus pentosus strains isolated from biofilms on the skin of green table olives are presented here. These genome sequences will assist in revealing the potential probiotic properties of these strains, as the intake of fermented olives implicates the passage of millions of Lactobacillus spp. throughout a consumer's gastrointestinal tract.

Microbial biogeography of Spanish-style green olive fermentations in the province of Seville, Spain

The aim of this study is to examine the biogeography of the microbial communities associated to the Spanish-style green olive fermentations in the province of Seville (Andalucía, south-western Spain). Also, to understand how microorganisms colonize and persist in non-sterile food fermentations across a specific table olive producing area, i.e. a specific “agroecosystem”. The microbial diversity, bacteria and yeast, in 30 ten-ton fermenters of three different fermentations yards (patios) along the olive fermentation was studied. A total of 951 microbial isolates were obtained which were clustered according to their RAPD profile. A total of 376 distinct genotypes were identified, belonging to 57 different microbial species, 41 bacterial and 16 yeast species. Up to 16 bacterial species had not been described before in table olives. Only the species Lactobacillus pentosus showed a ubiquitous presence in all 30 fermenters. Pediococcus parvulus, Lactobacillus collinoides/paracollinoides, Lactobacillus coryniformis, Lactobacillus plantarum, Pichia manshurica and Candida thaimueangensis were found in every patio. Cosmopolitan strains, up to 15, were shared by the three patios and belonged to the species L. pentosus (12 strains), P. parvulus (1), L. collinoides/paracollinoides (1) and P. manshurica (1). To expand our biodiversity analyses to the “regional” level, we have compared our results with those obtained from two previously studied patios of similar characteristics and in the same geographical area. PERMANOVA analysis of the microbial community composition revealed significant differences among different patios in their structure at every fermentation stage. In contrast, SIMPER analyses showed that, as fermentation progressed, the overall dissimilarities among patios were reduced. Discriminant species were identified for each fermentation stage. Among these, L. pentosus and P. parvulus were “eu-constant” species, while L. collinides/paracollinoides and Marinilactibacillus psychrotolerans group were “constant” species that could be considered microbial key taxa based on the occurrence stability index. The characteristic and, presumably, well adapted microbiota associated to the Spanish-style olive fermentations at the specific geographic area described here is a valuable natural resource which should be preserved conveniently. To our knowledge, this is the first study on the microbial biogeography of table olive fermentations, both at the species and strain levels.

The use of starter cultures in the table olive fermentation can modulate the antiadhesive properties of brine exopolysaccharides against enterotoxigenic Escherichia coli.

The present study aimed to evaluate different mates of Candida boidinii and Lactobacillus pentosus strains as starters in green table olive fermentation. Changes in fermentation characteristics as well as changes in the functional properties of the microbial exopolysaccharides (EPS) produced during the process were registered. The in vitro adhesion test demonstrated that most EPS samples could specifically attach ETEC K88. In vitro studies with porcine intestinal cells showed the improved blocking activity of the fimbria (blocking test) when the mutant strain L. pentosus 119-14MT was used alone as a starter. All EPS samples showed the ability to block receptors in the cells (exclusion test) although without differences between starter treatments. In the displacement test, EPS samples failed to remove the pathogen once attached. According to these results, L. pentosus 119-14MT, a high EPS variant, seemed to be the most effective starter improving the anti-adhesive properties of brine EPS and increasing its ability to block the ETEC K88 fimbria. These results illustrate that the anti-adhesive properties of the EPSs produced during the traditional fermentation of olives could be modulated by the use of defined starters. This opens the door to new fermentation processes aimed to produce green table olives as functional food to prevent ETEC diarrhea.

Approaches to improve the growth of the starter lactic acid bacterium OM13 during the early stages of green Spanish-style table olive production

The present research aimed at determining the optimal conditions for the lactic acid fermentation of green Spanish-style table olives. The work is a follow-up, and focuses on the performance of the commercial starter strain Lactobacillus pentosus OM13 by applying an acclimatization step and the addition of nutrients, and concentrations of lactic acid that were previously investigated. The acclimatization of the cells warranted the dominance of the starter culture even at an inoculation level of 2 Log cycles lower than that commonly used in standard fermentation. A significant effect was found in terms of acidification kinetics within the first week of fermentation, with the highest decrease in pH, at ~2.5 units, which occurred in the trial and after inoculation with 106 CFU/mL of starter after acclimatation (EO3) that showed values similar to control C obtained with Lactobacillus pentosus OM13 at a concentration of 107 CFU/mL. After day 3, further decreases in pH of up to 4.30 were observed until day 30, and then these values remained almost constant until the end of process (day 190) when lower pH values were reached for trial EO3 and control C. The results of microbiological dynamics, the changes in VOCs and finally the effect of the processes on the sensory analysis of the fermented product were investigated by multivariate analysis. The acclimatization process and the initial inoculation level influenced the bacterial microflora, aromatic compounds and organoleptic characteristics of the final product. Finally, the trials C, EO2 and EO3 showed higher values (60-80%) of preferences and satisfaction compared to other experimental productions.

Effect of Spanish-style processing steps and inoculation with Lactobacillus pentosus starter culture on the volatile composition of cv. Manzanilla green olives

The effects of the main steps of Spanish-style processing (alkaline treatment and fermentation) on the volatile composition of cv. Manzanilla green olives were studied. Both spontaneous and controlled fermentations were considered. In the latter case, a Lactobacillus pentosus strain from green olive fermentation brine was used as starter culture. The volatile profile was determined by headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC–MS). Most of the volatile compounds detected in fresh olives decreased or were undetected after alkaline treatment, while several compounds (mostly acetic acid, 2-methylbutanoic acid, and ethanol) were formed as a result of this treatment. Over 50 new volatile components, mostly esters and phenols, appeared as a result of fermentation. The most outstanding finding was a considerable increase in 4-ethyl phenol (almost 100-fold increase) in inoculated olives compared to the uninoculated product. However, a sensory panel did not find significant differences in odor perception.

Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-β Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.

Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells.

This study aims to explore the biological functions of the isolated exopolysaccharides (EPSs) produced during the industrial fermentation of olives against enterotoxigenic E. coli (ETEC) K88. Exopolysaccharides were isolated from five industrial fermenters. Analysis of their monosaccharide composition by GLC revealed that the main components were glucose (27%-50%) and galactose (23%-33%) followed by rhamnose (4-23%) and arabinose (6-17%). The 1H NMR spectrum showed a very similar profile between samples, and a more in-depth analysis revealed the presence of an α-pyranose in the form of α-d-Glcp-(1→) and two different α-furanoses, with chemicals shift values, suggesting the presence of α-d-Glcf and α-d-Galf. Miniaturized in vitro tests demonstrated the ability of EPS samples to attach specifically to ETEC K88 (P < 0.05) with variable intensities. The competition test did not show the ability to block the ETEC K88 adhesion to IPEC-J2 cells; however, in the displacement test, all EPS samples were shown to effectively remove the pathogens attached to the cells (P < 0.01). These results suggest that the EPSs produced during the fermentation of table green olives could interfere with the attachment of opportunistic pathogens onto the intestinal epithelial cells. This would open the possibility of novel functional properties for this traditional Mediterranean fermented food and for the isolated EPSs as candidates for nutraceutics to be used in human and/or animal diets in the prevention and treatment of ETEC diarrhoea.

Effects of Exogenous Yeast and Bacteria on the Microbial Population Dynamics and Outcomes of Olive Fermentations.

In this study, we examined Sicilian-style green olive fermentations upon the addition of Saccharomyces cerevisiae UCDFST 09-448 and/or Pichia kudriazevii UCDFST09-427 or the lactic acid bacteria (LAB) Lactobacillus plantarum AJ11R and Leuconostoc pseudomesenteroides BGM3R. Olives containing S.cerevisiae UCDFST 09-448, a strain able to hydrolyze pectin, but not P.kudriazevii UCDFST 09-427, a nonpectinolytic strain, exhibited excessive tissue damage within 4weeks. DNA sequencing of fungal internal transcribed spacer (ITS) regions and comparisons to a yeast-specific ITS sequence database remarkably showed that neither S.cerevisiae UCDFST 09-448 nor P.kudriazevii UCDFST 09-427 resulted in significant changes to yeast species diversity. Instead, Candida boidinii constituted the majority (>90%) of the total yeast present, independent of whether S.cerevisiae or P.kudriazevii was added. By comparison, Lactobacillus species were enriched in olives inoculated with potential starter LAB L.plantarum AJ11R and L.pseudomesenteroides BGM3R according to community 16S rRNA gene sequence analysis. The bacterial diversity of those olives was significantly reduced and resembled control fermentations incubated for a longer period of time. Importantly, microbial populations were highly dynamic at the strain level, as indicated by the large variations in AJ11R and BGM3R cell numbers over time and reductions in the numbers of yeast isolates expressing polygalacturonase activity. These findings show the distinct effects of exogenous spoilage and starter microbes on indigenous communities in plant-based food fermentations that result in very different impacts on product quality. IMPORTANCE Food fermentations are subject to tremendous selective pressures resulting in the growth and persistence of a limited number of bacterial and fungal taxa. Although these foods are vulnerable to spoilage by unintended contamination of certain microorganisms, or alternatively, can be improved by the deliberate addition of starter culture microbes that accelerate or beneficially modify product outcomes, the impact of either of those microbial additions on community dynamics within the fermentations is not well understood at strain-specific or global scales. Herein, we show how exogenous spoilage yeast or starter lactic acid bacteria confer very different effects on microbial numbers and diversity in olive fermentations. Introduced microbes have long-lasting consequences and result in changes that are apparent even when levels of those inoculants and their major enzymatic activities decline. This work has direct implications for understanding bacterial and fungal invasions of microbial habitats resulting in pivotal changes to community structure and function.

Enhancement of the Knowledge on Fungal Communities in Directly Brined Aloreña de Málaga Green Olive Fermentations by Metabarcoding Analysis.

Nowadays, our knowledge of the fungal biodiversity in fermented vegetables is limited although these microorganisms could have a great influence on the quality and safety of this kind of food. This work uses a metagenetic approach to obtain basic knowledge of the fungal community ecology during the course of fermentation of natural Aloreña de Málaga table olives, from reception of raw material to edible fruits. For this purpose, samples of brines and fruits were collected from two industries in Guadalhorce Valley (Málaga, Spain) at different moments of fermentation (0, 7, 30 and 120 days). The physicochemical and microbial counts performed during fermentation showed the typical evolution of this type of processes, mainly dominated by yeasts in apparent absence of Enterobacteriaceae and Lactobacillaceae. High-throughput barcoded pyrosequencing analysis of ITS1-5.8S-ITS2 region showed a low biodiversity of the fungal community, with the presence at 97% identity of 29 different fungal genera included in 105 operational taxonomic units (OTUs). The most important genera in the raw material at the moment of reception in the industry were Penicillium, Cladosporium, Malassezia, and Candida, whilst after 4 months of fermentation in brines Zygotorulaspora and Pichia were predominant, whereas in fruits were Candida, Penicillium, Debaryomyces and Saccharomyces. The fungal genera Penicillium, Pichia, and Zygotorulaspora were shared among the three types of substrates during all the course of fermentation, representing the core fungal population for this table olive specialty. A phylogenetic analysis of the ITS sequences allowed a more accurate assignment of diverse OTUs to Pichia manshurica, Candida parapsilosis/C. tropicalis, Candida diddensiae, and Citeromyces nyonensis clades. This study highlights the existence of a complex fungal consortium in olive fermentations including phytopathogenic, saprofitic, spoilage and fermentative genera. Insights into the ecology, identification and quantification of fungi species in olive fermentation will facilitate the design of new strategies to improve the quality and safety of this fermented vegetable.

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.

Controlled fermentation of Moroccan picholine green olives by oleuropein-degrading Lactobacilli strains

The control of the spontaneous fermentation process of un-debittered Moroccan Picholine green olives was undertaken basing the inoculation with two lactobacilli strains ( Lactobacillus plantarum S175 and Lactobacillus pentosus S100). These strains, previously selected in our laboratory for their oleuropein-degrading capacity, were inoculated in olives brined at 5% of NaCl, and then incubated at 30 AaC. The physico-chemical parameters (pH, free acidity, reducing sugars, sodium chloride, oleuropein and its hydrolysis products), and the microbiological parameters (mesophilic aerobic bacteria, coliforms, Staphylococcus , lactic acid bacteria and yeasts and moulds), were regularly analyzed during the fermentation time. The results obtained showed the effectiveness of the lactic acid bacteria strains to develop suitable oleuropein biodegradation and controlled lactic fermentation processes more than the un-inoculated olives (control). This result was confirmed by the rapid elimination of coliforms and staphylococcus, the accumulation of hydroxytyrosol as a result of oleuropein biodegradation, and a drastic reduction in spoiled olives with good quality fermented olives.

Technological Potential of Lactobacillus Strains Isolated from Fermented Green Olives: In Vitro Studies with Emphasis on Oleuropein-Degrading Capability.

Technological properties of two strains of Lactobacillus plantarum (B3 and B11) and one of Lactobacillus pentosus (B4), previously isolated from natural fermented green olives, have been studied in vitro. Acidifying ability, salt, temperature, and pH tolerances of all strains were found in the range reported for similar strains produced in Italy and optimal growth conditions were found to be 6.0–8.0 pH, 15–30°C temperature, and less than 6% NaCl. Moreover, all strains showed very good tolerance to common olive phenol content (0.3% total phenol) and high oleuropein-degrading capability. It was found that medium composition affected the bacterial oleuropein degradation. B11 strain grown in a nutrient-rich medium showed a lower oleuropein-degrading action than when it was cultivated in nutrient-poor medium. Furthermore, enzymatic activity assays revealed that oleuropein depletion did not correspond to an increase of hydroxytyrosol, evidencing that bacterial strains could efficiently degrade oleuropein via a mechanism different from hydrolysis.

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.

Survival of potential probiotic lactic acid bacteria on fermented green table olives during packaging in polyethylene pouches at 4 and 20 °C

The survival of selected lactic acid bacteria (LAB) with in vitro probiotic potential was studied during storage of cv. Halkidiki green olives previously subjected to inoculated Spanish-style fermentation. After fermentation olives were packed in polyethylene pouches, covered with freshly prepared brine (9%, w/v, NaCl), acidified with 2‰ (w/v) citric acid and 1.5‰ (w/v) ascorbic acid, and stored at 4 and 20 °C for 357 days. Four packing treatments were studied, namely olives previously fermented by (i) the indigenous microbiota (control); (ii) Lactobacillus pentosus B281; (iii) Lactobacillus plantarum B282; and (iv) a co-culture of both LAB strains. Microbiological analyses were performed on the olives in parallel with physicochemical changes (pH, titratable acidity, salt content, aw and colour) at the early (day 1), middle (day 197) and final stage (day 357) of storage, as well as sensory evaluation at the end of the storage. The survival of probiotic strains was confirmed by Pulsed Field Gel Electrophoresis (PFGE). LAB decreased throughout storage reaching a final population of ca. 3.5–4.0 log CFU/g and 4.5–5.0 log CFU/g at 4 and 20 °C, respectively. The pH values ranged between 3.90 and 4.61 during storage depending on packaging condition. PFGE analysis revealed that L. pentosus B281 and L. plantarum B282 showed a high survival rate with a recovery of 100 and 96%, respectively, at 4 °C, and less than 20% for both strains at 20 °C. Finally, in the packing treatment with a co-culture of both strains, L. pentosus dominated over L. plantarum throughout storage at both temperatures.

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.

Quantification and characterization of microbial biofilm community attached on the surface of fermentation vessels used in green table olive processing

The aim of the present study was the quantification of biofilm formed on the surface of plastic vessels used in Spanish-style green olive fermentation and the characterization of the biofilm community by means of molecular fingerprinting. Fermentation vessels previously used in green olive processing were subjected to sampling at three different locations, two on the side and one on the bottom of the vessel. Prior to sampling, two cleaning treatments were applied to the containers, including (a) washing with hot tap water (60°C) and household detergent (treatment A) and (b) washing with hot tap water, household detergent and bleach (treatment B). Population (expressed as logCFU/cm2) of total viable counts (TVC), lactic acid bacteria (LAB) and yeasts were enumerated by standard plating. Bulk cells (whole colonies) from agar plates were isolated for further characterization by PCR-DGGE. Results showed that regardless of the cleaning treatment no significant differences were observed between the different sampling locations in the vessel. The initial microbial population before cleaning ranged between 3.0–4.5logCFU/cm2 for LAB and 4.0–4.6logCFU/cm2 for yeasts. Cleaning treatments exhibited the highest effect on LAB that were recovered at 1.5logCFU/cm2 after treatment A and 0.2logCFU/cm2 after treatment B, whereas yeasts were recovered at approximately 1.9logCFU/cm2 even after treatment B. High diversity of yeasts was observed between the different treatments and sampling spots. The most abundant species recovered belonged to Candida genus, while Wickerhamomyces anomalus, Debaryomyces hansenii and Pichia guilliermondii were frequently detected. Among LAB, Lactobacillus pentosus was the most abundant species present on the abiotic surface of the vessels.

Determination of triterpenic acids in natural and alkaline-treated Greek table olives throughout the fermentation process

Maslinic and oleanolic acids are among the most abundant triterpenic acids found in olive fruits. Both acids are considered to be important bioactive compounds which can confer multiple beneficial health effects to the consumer. In the present work, we have monitored and quantified maslinic and oleanolic acids throughout processing in alkaline-treated green olives (Spanish-style) and in natural green olives of the Conservolea variety that is particularly popular in Greece. Our findings clearly demonstrate that the fast de-bittering process with NaOH treatment in Spanish-style olives has a profound negative effect on the concentration of both acids. This decrease of concentration was more prominent regarding maslinic acid when compared to oleanolic acid. In contrast, the slow de-bittering during natural fermentation of green olives had no effect on the content of maslinic or oleanolic acid. To verify the broad applicability of our observation we have also looked into the natural processing of the Kalamon variety (Greek-style). Our findings were consistent, since once again, natural fermentation did not influence the concentration of both acids.

Microbial diversity and dynamics of Spanish-style green table-olive fermentations in large manufacturing companies through culture-dependent techniques

We have studied the microbiota associated to Spanish-style green olive fermentations, attending to its dynamics along the time. Twenty 10-tonne fermenters were selected from two large table-olive manufacturing companies in southern Spain. While culture-dependent methodology was used to isolate the microorganisms, molecular methods were used to identify the isolates. A total of 1070 isolates were obtained, resulting in 929 bacterial and 141 yeast isolates. Thirty seven different bacterial species were isolated, belonging to 18 different genera, while 12 yeast species were isolated, belonging to 7 distinct genera. This fermentation was dominated by the species Lactobacillus pentosus, while its accessory microbiota was variable and depended on the fermentation stage and the actual fermentation yard (“patio”). It was noticeable the abundance of lactic acid bacteria isolates, belonging to 16 different species. Twenty bacterial species were isolated for the first time from Spanish-style green olive fermentations, while 17 had not been described before in any table olive preparation. The genera Brachybacterium, Paenibacillus, Sporolactobacillus, Paracoccus and Yersinia had not been cited before from any table olive preparation. Saccharomyces cerevisiae and Candida thaimueangensis appeared to dominate the yeast microbiota. Candida butyri/asseri and Rhodotorula mucilaginosa had not been described before from Spanish-style preparations, while Saturnispora mendoncae was isolated for the first time from any table olive preparation. Biodiversity was analysed through different alpha and beta indexes which showed the evolution of the microbiota over time.