Impact of lactic acid bacteria inoculation on fungal diversity during Spanish-style green table olive fermentations.

This study delved into the evolution of fungal population during the fermentation of Spanish-style green table olives (Manzanilla cultivar), determining the influence of different factors such as fermentation matrix (brine or fruit) or the use of a lactic acid bacteria inoculum, on its distribution. The samples (n = 24) were directly obtained from industrial fermentation vessels with approximately 10.000 kg of fruits and 6.000 L of brines. Our findings showcased a synchronized uptick in lactic acid bacteria counts alongside fungi proliferation. Metataxonomic analysis of the Internal Transcribed Spacer (ITS) region unearthed noteworthy disparities across different fermentation time points (0, 24, and 83 days). Statistical analysis pinpointed two Amplicon Sequence Variants (ASV), Candida and Aureobasidium, as accountable for the observed variances among the different fermentation time samples. Notably, Candida exhibited a marked increase during 83 days of fermentation, opposite to Aureobasidium, which demonstrated a decline. Fungal biodiversity was slightly higher in brines than in fruits, whilst no effect of inoculation was noticed. At the onset of fermentation, prominently detected genera were also Mycosphaerella (19.82 %) and Apohysomyces (16.31 %), hitherto unreported in the context of table olive processing. However, their prevalence dwindled to nearly negligible levels from 24th day fermentation onwards (<2 %). On the contrary, they were replaced by the fermentative yeasts Saccharomyces and Isstachenkia. Results obtained in this work will be useful for designing new strategies for better control of table olive fermentations.

Functional features of the exopolysaccharide extracts produced by Lactiplantibacillus strains isolated from table olives.

This study evaluates the functional characteristics of the exopolysaccharide (EPS) extracts produced by various strains of Lactiplantibacillus pentosus (LPG1, 119, 13B4, and Lp13) and Lactiplantibacillus plantarum (Lp15) isolated from table olives. None of the EPS crude extracts showed cytotoxicity when administered to THP-1 human macrophage cells at dosages ranging from 6.25 to 50 µg mL-1. Many exhibited anti-inflammatory properties (reduction of pro-inflammatory cytokines TNF-α and IL-6 production) and antioxidant activity (reduction of ROS%) when macrophages were stimulated with Escherichia coli lipopolysaccharide. Notably, the EPS extract produced by the L. pentosus LPG1 strain had the best results corroborated by western blot immune analysis for differential expression of COX-2, Nrf-2, and HO-1 proteins, with the most significant antioxidant and anti-inflammatory response observed at a dosage of 50 µg mL-1. Chemical analysis revealed that the EPS extract produced by this strain contains a heteropolymer composed of mannose (35.45%), glucose (32.99%), arabinose (17.93%), xylose (7.48%), galactose (4.03%), rhamnose (1.34%), and fucose (0.77%). Finally, we conducted response surface methodology to model the EPS extract production by L. pentosus LPG1 considering pH (3.48-8.52), temperature (16.59-33.41 °C) and salt concentration (0.03-8.77% NaCl) as independent variables. The model identified linear effects of salt and pH and quadratic effects of salt as significant terms. The maximum EPS extract production (566 mg L-1) in a synthetic culture medium (MRS) was achieved at pH 7.5, salt 7.0%, and a temperature of 20 °C. These findings suggest the potential for novel applications for the EPS produced by L. pentosus LPG1 as nutraceutical candidates for use in human diets.

Tracking Microbial Diversity and Hygienic-Sanitary Status during Processing of Farmed Rainbow Trout (Oncorhynchus mykiss).

Aquaculture is becoming a strategic sector for many national economies to supply the increasing demand for fish from consumers. Fish culture conditions and processing operations can lead to an increase in microbial contamination of farmed fish that may shorten the shelf-life of fish products and byproducts, and ready-to-eat fishery products. The objective of this study was to evaluate the hygienic-sanitary status of water, environment, and processing of fresh-farmed rainbow trout (Oncorhynchus mykiss) fillets produced in a local fish farm in Andalusia, Spain. To achieve this, a longitudinal study was carried out by collecting environmental (air and food-contact surfaces), water from fish ponds, and rainbow trout samples. Thereby, seven sampling visits were performed between February 2021 and July 2022, where foodborne pathogens and spoilage microorganisms, together with physicochemical parameters, were analysed in the collected samples. Further, microbial identification of microbiota was achieved through a culture-dependent technique using blast analysis of 16S RNA gene sequencing. The results showed that Listeria monocytogenes and Salmonella were not detected in the analysed samples. Regarding the hygienic-sanitary status of the fish farm, the slaughtering bath, the eviscerating machine and the outlet water from fish ponds presented the highest counts of coliforms, Enterobacteriaceae, and Aerobic Mesophilic Bacteria. Staphylococcus aureus and sulphite-reducing Clostridium were identified in the conveyor belts, fish flesh, and viscera. The 16S RNA identification confirmed the presence of viable spoilage bacteria such as Citrobacter gillenii, Macrococcus caseolyticus, Hafnia paralvei, Lactococcus lactis, Lactococcus cremoris, Klebsiella, Escherichia coli, Morganella morganii, and Shewanella. Three of these genera (Citrobacter, Hafnia, and Pseudomonas) were present in all types of samples analysed. The results evidenced potential transmission of microbial contamination from contaminated packaging belts and boxes, evisceration and filleting machines to flesh and viscera samples, thus the establishment of control measures should be implemented in fish farm facilities to extend the shelf-life of farmed fishery products.

Fungal biodiversity in commercial table olive packages.

In this work, a total of 72 commercial table olive packages obtained from different international markets were analysed to determine their fungal biodiversity. Viable fungal counts ranged from the detection threshold (<1.6 log10 CFU/g in 25% of cases) to a maximum of 5.86 log10 CFU/g. Assignation of fungal taxonomy was carried out through a metataxonomic analysis of the ITS region, which revealed that almost half of the total sequences obtained from all packages corresponded to the Pichia genus (44.08%), followed by Citeromyces (14.45%), Candida (8.07%), and Wickerhamomyces (6.95%). In lower proportions were also detected other genera such as Starmerella (3.60%), Saccharomyces (2.24%), Debaryomyces (2.08%), and Dekkera (2.05%). The statistical analysis allowed to link certain taxa to specific types of elaboration (lye treated, green, and black natural olives), presentation (pitted, whole, or sliced samples), and packaging material/system (glass, PET, plastic bags, and vacuum). Likewise, Zygotorulaspora genus was especially sensitive to the presence of potassium sorbate, while other genera such as Sporobolomyces, Moniliella, and Gibellulopsis were more abundant in packages treated with this preservative. Lastly, potential pathogenic fungal genera such as Alternaria, Kodamaea, Lodderomyces, Malasessia, or Aspergillus were detected in low proportions (<0.3%), although with higher representation in some individual samples. Our results contribute to improving our knowledge of the fungal population associated with this ready-to-eat fermented vegetable, providing us a strong tool to assess the safety, stability, and quality of the final product.

Delving into the bacterial diversity of spoiled green Manzanilla Spanish-style table olive fermentations.

This work applies metataxonomic, standard statistics, and compositional data (CoDa) techniques to study the bacterial diversity of spoiled and normal Spanish-style table olive fermentations, analysing a total of 10-tons of industrial fermentation containers from two processing yards. Forty percent were affected by butyric, sulfidic, or putrid spoilage, while 60% followed the ordinary fermentation course. The samples were obtained at 30 days of fermentation, determining their 16S rRNA gene Amplicon Sequence Variant compositions (ASVs). The butyric containers showed a bacterial profile strongly associated with the genera Enterococcus, Leuconostoc, and Atlantibacter, but also with Lactiplantibacillus and Melissococcus, and less confident to Raoultella, Enterobacter, Serratia, and Celerinatantimonas. The sulfidic fermentation was linked to Alkalibacterium and, to a lesser extent, Marinilactibacillus and the absence of Lactiplantibacillus. Putrid spoilage was mainly related to Halolactibacillus and Alkalibacterium. Sulfidic/putrid (together) differed from butyric spoilage by the presence of Alkalibacterium/Marinilactibacillus as well as by Halomonas/Halanaerobium. Lactiplantibacillus dominated normal fermentations, but Vibrio was also frequently found (0-46%), apparently not causing any alteration. These results contribute to a better microbial characterisation of non-zapatera spoiled table olive fermentations. They also suggest using several statistical techniques to discriminate normal vs spoiled fermentations adequately.

New Insights into Microbial Diversity of the Traditional Packed Table Olives Aloreña de Málaga through Metataxonomic Analysis.

Aloreña de Málaga is a table olive especially characterised by its natural freshness and short shelf-life. In this work, we applied a metataxonomic approach to unravel the microbial diversity of bacterial and fungi populations through the shelf-life of traditionally packed Aloreña de Málaga. A significant increase in lactic acid bacteria and mesophilic aerobic populations was observed during shelf-life, reaching the maximum population levels (4-5 log10 CFU) at the end of the study (260 days). On the contrary, a rapid reduction in yeast and mould populations was reported. The use of a metataxonomic analysis based on the amplification of 16S (bacteria) and internal transcribed spacer (ITS) region (fungi) regions revealed a low diversity for both microbial groups. Lactiplantibacillus (65.05 ± 8.65% in brine vs. 58.70 ± 15.70% in fruit), Pediococcus (28.17 ± 7.36% in brine vs. 27.20 ± 15.95% in fruit), and Celerinatantimonas (4.64 ± 1.08% in brine vs. 11.82 ± 18.17% in fruit) were the main genera found among bacteria, and an increase in Lactiplantibacillus and a reduction in Celerinatantimonas populations during the shelf-life were observed. On the other hand, Citeromyces was the dominant fungi genus (54.11 ± 2.00% in brine vs. 50.91 ± 16.14% in fruit), followed by Candida (8.80 ± 2.57% in brine vs. 12.32 ± 8.61% in fruit) and Penicillium (6.48 ± 1.87% vs. 8.48 ± 4.43% in fruit). No food-borne pathogen genera were detected in any of the samples analysed, indicating the high level of food safety found in this ready-to-eat fermented vegetable. Data obtained in this work will help in the design of new strategies for the control of microbial populations during the shelf-life of Aloreña de Málaga.

Exploring bacteria diversity in commercialized table olive biofilms by metataxonomic and compositional data analysis.

In this work, a total of 72 samples of non-thermally treated commercial table olives were obtained from different markets of the world. Then, prokaryotic diversity in olive biofilms was investigated by metataxonomic analysis. A total of 660 different OTUs were obtained, belonging to Archaea (2.12%) and Bacteria domains (97.88%). From these, 41 OTUs with a proportion of sequences ≥ 0.01% were studied by compositional data analysis. Only two genera were found in all samples, Lactobacillus, which was the predominant bacteria in the biofilm consortium (median 54.99%), and Pediococcus (26.09%). Celerinatantimonas, Leuconostoc, Alkalibacterium, Pseudomonas, Marinilactibacillus, Weissella, and the family Enterobacteriaceae were also present in at least 80% of samples. Regarding foodborne pathogens, only Enterobacteriaceae, Vibrio, and Staphylococcus were detected in at least 91.66%, 75.00%, and 54.10% of samples, respectively, but their median values were always below 0.15%. Compositional data analysis allowed discriminating between lye treated and natural olive samples, as well as between olives packaged in glass, PET and plastic bags. Leuconostoc, Celerinatantimonas, and Alkalibacterium were the bacteria genera with a higher discriminant power among samples. These results expand our knowledge of the bacteria diversity in olive biofilms, providing information about the sanitary and hygienic status of this ready-to-eat fermented vegetable.

Influence of Acid Adaptation on the Probability of Germination of Clostridium sporogenes Spores Against pH, NaCl and Time.

The Clostridium sp. is a large group of spore-forming, facultative or strictly anaerobic, Gram-positive bacteria that can produce food poisoning. The table olive industry is demanding alternative formulations to respond to market demand for the reduction of acidity and salt contents in final products. while maintaining the appearance of freshness of fruits. In this work, logistic regression models for non-adapted and acid-adapted Clostridium sp. strains were developed in laboratory medium to study the influence of pH, NaCl (%) and time on the probability of germination of their spores. A Clostridium sporogenes cocktail was not able to germinate at pH < 5.0, although the adaptation of the strains produced an increase in the probability of germination at 5.0-5.5 pH levels and 6% NaCl concentration. At acidic pH values (5.0), the adapted strains germinated after 10 days of incubation, while those which were non-adapted required 15 days. At pH 5.75 and with 4% NaCl, germination of the adapted strains took place before 7 days, while several replicates of the non-adapted strains did not germinate after 42 days of storage. The model was validated in natural green olive brines with good results (>81.7% correct prediction cases). The information will be useful for the industry and administration to assess the safety risk in the formulation of new processing conditions in table olives and other fermented vegetables.

Foodborne Pathogen Survival in Commercial Aloreña de Málaga Table Olive Packaging.

This study presents an approach to determine the survival of diverse foodborne pathogens (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica) in three Aloreña de Málaga table olive commercial presentations (fresh green, traditional, and cured olives). The microbial survival in this green natural table olive speciality was fit using a log-linear regression model implemented in GInaFIT. The contents of sugars, phenolic compounds, additives, salt, pH, and levels of autochthonous microorganisms differed among presentations and affected the survival of microorganisms. The inoculated initial populations of pathogens (7-8 log10 CFU/mL brine) decreased rapidly and, 48 h after inoculation, their counts were always below the detection limit (<1.3 log10 CFU/mL), except for S. aureus in the fresh green presentation which was ∼ 5.0 log10 CFU/mL. The highest maximum death rates (kmax ) and lowest periods for 4 log10 reductions (4Dr) were observed in cured olives but decreased and increased, respectively, from the traditional to the fresh green presentations. L. monocytogenes and S. aureus were the most resistant species. The multivariate analysis showed that high concentrations of compounds released from the olives (sugars and phenols) were positively associated to 4Dr and negatively to kmax . Conversely, the presence of preservatives reduced 4Dr. This study, executed in commercial products, pointed out that packaged table olives are adverse habitats for foodborne pathogens with their effects being presentation dependent. The survival of S. aureus was particularly long in green fresh Aloreña de Málaga table olives packaged without preservatives; therefore, its changes in this presentation still requires further in-deep research.

Reduction of the Bitter Taste in Packaged Natural Black Manzanilla Olives by Zinc Chloride.

The work assays the use of various concentrations of ZnCl2 (0.0-0.1%, w/v) in packaged natural black Manzanilla table olives. The transformations were followed for 4 months. The presence of Zn modified the leaching of total sugars (sucrose, glucose, fructose, and mannitol) into the brine, which decreased as the ZnCl2 content increased. Over the study, sucrose and glucose were exhausted while fructose, although consumed, left some final residues and the use of mannitol was limited. Titratable acidity was always gradually formed causing the subsequent pH decrease, which stabilized at ≈3.5. Acetic and mainly lactic acid were also formed during the assay, reaching the highest level of lactic acid in the 0.050% ZnCl2 treatment, followed by the Control. The acids were formed by lactic acid bacteria (LAB) (Lactobacillus pentosus, 39%, and Lactobacillus plantarum, 61%). However, the most outstanding Zn effect was found on the olive sensory characteristics: its presence markedly reduced the bitter notes, increased the overall appreciation, and the treatment containing 0.075% ZnCl2 had the highest scores in hardness, crunchiness, and overall appreciation. Therefore, the addition of ZnCl2 into packaged natural table olives may lead to healthy products with desirable sensory characteristics which, in turn, could promote consumption.

A Probabilistic Decision-Making Scoring System for Quality and Safety Management in Aloreña de Málaga Table Olive Processing.

Table olives are one of the most representatives and consumed fermented vegetables in Mediterranean countries. However, there is an evident lack of standardization of production processes and HACCP systems thus implying the need of establishing decision-making tools allowing their commercialization and shelf-life extension. The present work aims at developing a decision-making scoring system by means of a probabilistic assessment to standardize production process of Aloreña de Málaga table olives based on the identification of potential hazards or deficiencies in hygienic processes for the subsequent implementation of corrective measures. A total of 658 microbiological and physico-chemical data were collected over three consecutive olive campaigns (2014-2016) to measure the variability and relative importance of each elaboration step on total hygienic quality and product safety. Three representative companies were visited to collect samples from food-contact surfaces, olive fruits, brines, air environment, olive dressings, water tanks, and finished/packaged products. A probabilistic assessment was done based on the establishment of Performance Hygiene and Safety Scores (PHSS 0-100%) through a standardized system for evaluating product acceptability. The mean value of the global PHSS for the Aloreña de Málaga table olives processing (PHHSFTOT) was 64.82% (90th CI: 52.78-76.39%) indicating the high variability among facilities in the evaluated processing steps on final product quality and safety. Washing and cracking, and selection and addition of olive dressings were detected as the most deficient ones in relation to PHSSFi values (p < 0.05) (mean = 53.02 and 56.62%, respectively). The relative contribution of each processing step was quantified by different experts (n = 25) from the Aloreña de Málaga table olive sector through a weighted PHSS (PHSSw). The mean value of PHSSw was 65.53% (90th CI: 53.12-77.52%). The final processing steps obtained higher values for PHSSw being the finished product the most relevant one (mean = 18.44%; 90% CI: 10.34-25.33%). Sensitivity analysis concluded that intervention measures focused on reducing the contamination of washing brines could lead to an improvement of PHSSFTOT value to 67.03%. The present work can be potentially applied in the Aloreña de Málaga table olive food sector for improving food quality and safety assurance.

Microbiological and Physicochemical Changes in Natural Green Heat-Shocked Aloreña de Málaga Table Olives.

Preserving the highly appreciated natural freshness of Aloreña de Málaga table olives and preventing their progressive darkening during processing is a major challenge. In this work, heat-shocked (60°C, 5 min) fruits were processed according to the three denominations referred to in the Protected Designation of Origen (cured, fresh green, and traditional) and their characteristics compared with those that followed the habitual industrial process (controls). The results revealed that the effects of the heat treatment on the evolution of pH, titratable acidity, salt, sugar, organic acid, ethanol content, texture, and color of fruits as well as on microbial populations (yeasts and lactic acid bacteria) were slight in the case of the fresh green and cured presentations. However, the differences between heat-shocked and its control were remarkable in the traditional process. Notably, the heat treatment favored lactic acid fermentation, retention of the green appearance of the fruits, stability during packaging, and led to the highest sensory evaluation. The metagenomic analysis carried out at the end of the fermentation revealed the presence in all samples of three genera (Lactobacillus, Pediococcus, and Celerinatantimonas) which encompassed most of the sequences. The number of Lactobacillus sequences was statistically higher (p ≥ 0.05) in the case of traditional heat-shocked fruits than in its control.

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.

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.

In silico Logistic Model for Table Olive Related Microorganisms As a Function of Sodium Metabisulphite, Cinnamaldehyde, pH, and Type of Acidifying Agent.

A probabilistic/logistic model, based on binary data (growth/no growth), was used to assess the effects of sodium metabisulphite (SM) and cinnamaldehyde (CIN; 0-1000 mg/L) against the main microbial groups found in table olive environment [lactic acid bacteria (LAB), yeasts, and Enterobacteriaceae], according to pH (range 3.5-5.0), and type of acidifying agent (HCl or pyruvic acid). The inhibitory effect of SM depended on the pH while that of CIN was scarcely influenced by it (except for LAB). LAB were more sensitive to SM, while yeasts were to CIN. The use of pyruvic acid for correction of pH always produced a reduction (compared to HCl) of the inhibitory power of both preservatives. The in silico models for HCl showed that, at pH 4.0, and growth probability 0.01, the LAB population might be inhibited by the presence in the medium of 150 mg/L SM or 1000 mg/L CIN, while in the case of yeasts, 450 mg/L SM, or 150 mg/L CIN are required. No growth of Enterobacteriaceae was observed at this (or lower) pH level. The results obtained may contribute to the stabilization of non-thermally treated table olive packaging.

Survival of foodborne pathogens in natural cracked olive brines.

This work reports the survival (challenge tests) of foodborne pathogen species (Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica) in Aloreña de Málaga table olive brines. The inhibitions were fit using a log-linear model with tail implemented in GInaFIT excel software. The olive brine had a considerable inhibitory effect on the pathogens. The residual (final) populations (Fp) after 24 h was below detection limit (<1.30 log10 cfu/mL) for all species assayed. The maximum death rate (kmax) was 9.98, 51.37, 38.35 and 53.01 h(-1), while the time for 4 log10 reductions (4Dr) was 0.96, 0.36, 0.36 and 0.24 h for E. coli, S. aureus, L. monocytogenes and S. enterica, respectively. Brine dilutions increased Fp and 4Dr, while decreased kmax. A cluster analysis showed that E. coli had an overall quite different behaviour being the most resistant species, but the others bacteria behaved similarly, especially S. aureus and S. enterica. Partial Least Squares regression showed that the most influential phenols on microbial survival were EDA (dialdehydic form of decarboxymethyl elenolic acid), HyEDA (EDA linked to hydroxytyrosol), hydroxytyrosol 4-glucoside, tyrosol, and oleoside 11-methyl ester. Results confirm the adverse habitats of table olives for foodborne pathogenic microorganisms.

Effect of green Spanish-style Manzanilla packaging conditions on the prevalence of the putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2.

This work focuses on the persistence of the putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2 on green Spanish-style Manzanilla olives according to different packaging conditions and storage temperatures. The lactic acid bacteria population decreased with time but the highest survival counts (and lowest yeasts) at the end of storage (8 months) were observed in plastic pouches under nitrogen atmosphere and glass jars with brine stored at 20°C. Molecular techniques showed a 100% presence of the putative probiotic bacteria in biofilms adhered to olive epidermis, while it was absent in PPB (plastic pouches with brine) and in olives stored at 7°C. No changes in NaCl, pH or combined acidity were observed during the storage except for a slight increase in titratable acidity at 20°C. The color of the fruits was stable but degraded at 20°C for olives in plastic pouches with brine.

Evaluation and identification of poly-microbial biofilms on natural green Gordal table olives.

This work examines the formation of poly-microbial communities adhered to the epidermis of natural green Gordal olives and the application of different methodologies for recovery and counting of the microorganisms embedded in olive biofilms. The fermentation process was physicochemical and microbiologically monitored for 90 days, at which, formation of true biofilms on the skin of fermented fruits was confirmed by scanning electron microscopy. Then, samples of olives were taken and treated with sonication, enzymes, mechanical homogenization with stomacher and ultrasonic bath for biofilm disaggregation. The use of the stomacher for 1 min was the most effective treatment to release the lactic acid bacteria (6.6 log10 cfu g(-1)), whereas sonication for 5 min was the most efficient method for quantification of yeasts (up to 3.5 log10 cfu g(-1)). Molecular identification of isolates obtained from natural Gordal olive biofilms revealed that Lactobacillus pentosus was the only species found among lactic acid bacteria, while Pichia membranifaciens was the dominant yeast species, with higher counts obtained for the bacteria.

The effect of ZnCl2 on green Spanish-style table olive packaging, a presentation style dependent behaviour.

BACKGROUND: Zinc chloride has been used previously as a preservative in directly brined olives with promising results. However, this is the first time that the effects of ZnCl2 addition (0-1 g L(-1) ) on green Spanish-style table olive (cv. Manzanilla) packaging has been studied. RESULTS: The presence of ZnCl2 affected the physico-chemical characteristics of the products; the presence of the Zn led to lower pH values (particularly just after packaging) and titratable and combined acidity values than the control but did not produce clear trends in the colour parameters. No Enterobacteriaceae were found in any of the treatments evaluated. At the highest ZnCl2 concentrations, the lactic acid bacteria were inhibited while, unexpectedly, its presence showed a lower effect than potassium sorbate against the yeast population. Regardless of the use of potassium sorbate or ZnCl2 , the packages had a reduced microbial biodiversity because only Lactobacillus pentosus and Pichia galeiformis were found at the end of the shelf life. With respect to organoleptic characteristics, the presentations containing ZnCl2 were not differentiated from the traditional product. CONCLUSION: Zinc chloride was less efficient than potassium sorbate as a yeast inhibitor in green Spanish-style olives, showing clear presentation style dependent behaviour for this property. Its presence produced significant changes in chemical parameters but scarcely affected colour or sensory characteristics.

Survival of pathogenic and lactobacilli species of fermented olives during simulated human digestion.

The present survey uses a dynamic gastric and small intestinal model to assess the survival of one pathogenic (Escherichia coli O157:H7 EDL 933) and three lactobacilli bacteria with probiotic potential (Lactobacillus rhamnosus GG, L. pentosus TOMC-LAB2, and L. pentosus TOMC-LAB4) during their passage through the human gastrointestinal tract using fermented olives as the food matrix. The data showed that the survival of the E. coli strain in the stomach and duodenum was very low, while its transit through the distal parts (jejunum and ileum) resulted in an increase in the pathogen population. The production of Shiga toxins by this enterohemorrhagic microorganism in the ileal effluents of the in vitro system was too low to be detected by ELISA assays. On the contrary, the three lactobacilli species assayed showed a considerable resistance to the gastric digestion, but not to the intestinal one, which affected their survival, and was especially evident in the case of both L. pentosus strains. In spite of this, high population levels for all assayed microorganisms were recovered at the end of the gastrointestinal passage. The results obtained in the present study show the potential use of table olives as a vehicle of beneficial microorganisms to the human body, as well as the need for good hygienic practices on the part of olive manufacturers in order to avoid the possibility of contamination by food-borne pathogens.