Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

Microbiological Quality Estimation of Meat Using Deep CNNs on Embedded Hardware Systems.

Spectroscopic sensor imaging of food samples meta-processed by deep machine learning models can be used to assess the quality of the sample. This article presents an architecture for estimating microbial populations in meat samples using multispectral imaging and deep convolutional neural networks. The deep learning models operate on embedded platforms and not offline on a separate computer or a cloud server. Different storage conditions of the meat samples were used, and various deep learning models and embedded platforms were evaluated. In addition, the hardware boards were evaluated in terms of latency, throughput, efficiency and value on different data pre-processing and imaging-type setups. The experimental results showed the advantage of the XavierNX platform in terms of latency and throughput and the advantage of Nano and RP4 in terms of efficiency and value, respectively.

Rapid Assessment of Microbial Quality in Edible Seaweeds Using Sensor Techniques Based on Spectroscopy, Imaging Analysis and Sensors Mimicking Human Senses.

The expansion of the seaweed aquaculture sector along with the rapid deterioration of these products escalates the importance of implementing rapid, real-time techniques for their quality assessment. Seaweed samples originating from Scotland and Ireland were stored under various temperature conditions for specific time intervals. Microbiological analysis was performed throughout storage to assess the total viable counts (TVC), while in parallel FT-IR spectroscopy, multispectral imaging (MSI) and electronic nose (e-nose) analyses were conducted. Machine learning models (partial least square regression (PLS-R)) were developed to assess any correlations between sensor and microbiological data. Microbial counts ranged from 1.8 to 9.5 log CFU/g, while the microbial growth rate was affected by origin, harvest year and storage temperature. The models developed using FT-IR data indicated a good prediction performance on the external test dataset. The model developed by combining data from both origins resulted in satisfactory prediction performance, exhibiting enhanced robustness from being origin unaware towards microbiological population prediction. The results of the model developed with the MSI data indicated a relatively good prediction performance on the external test dataset in spite of the high RMSE values, whereas while using e-nose data from both MI and SAMS, a poor prediction performance of the model was reported.

Recent Advances and Applications of Rapid Microbial Assessment from a Food Safety Perspective.

Unsafe food is estimated to cause 600 million cases of foodborne disease, annually. Thus, the development of methods that could assist in the prevention of foodborne diseases is of high interest. This review summarizes the recent progress toward rapid microbial assessment through (i) spectroscopic techniques, (ii) spectral imaging techniques, (iii) biosensors and (iv) sensors designed to mimic human senses. These methods often produce complex and high-dimensional data that cannot be analyzed with conventional statistical methods. Multivariate statistics and machine learning approaches seemed to be valuable for these methods so as to "translate" measurements to microbial estimations. However, a great proportion of the models reported in the literature misuse these approaches, which may lead to models with low predictive power under generic conditions. Overall, all the methods showed great potential for rapid microbial assessment. Biosensors are closer to wide-scale implementation followed by spectroscopic techniques and then by spectral imaging techniques and sensors designed to mimic human senses.

Evolution of fungal community associated with ready-to-eat pineapple during storage under different temperature conditions.

The international market of fresh-cut products has witnessed dramatic growth in recent years, stimulated by consumer's demand for healthy, nutritious and convenient foods. One of the main challenging issues for the quality and safety of these products is the potential microbial spoilage that can significantly reduce their shelf-life. The complete identification of fresh-cut product microbiota together with the evaluation of environmental factors impact on microbial composition is of primary importance. We therefore assessed the fungal communities associated with the spoilage of ready-to-eat (RTE) pineapple using a metagenetic amplicon sequencing approach, based on the ITS2 region. Our results revealed a significant variability on fungal species composition between the different batches of RTE pineapple. The initial microbiota composition was the main influencing factor and determined the progress of spoilage. Temperature and storage time were the secondary factors influencing spoilage and their impact was depending on the initial prevalent fungal species, which showed different responses to the various modifications. Our results strongly suggest that further large-scale sampling of RTE pineapple production should be conducted in order to assess the full biodiversity range of fungal community involved in the spoilage process and for unravelling the impact of important environmental factors shaping the initial microbiota.

Spoilage potential of Bacillus subtilis in a neutral-pH dairy dessert.

The objective of this study was the characterization of the microbiota associated with spoilage of vanilla cream pudding during storage at different temperatures. Commercial cream samples were stored aerobically at 4, 8, 12 and 15 °C for a maximum time period of 40 days. At appropriate time intervals, cream samples were subjected to: (i) microbiological analyses, and (ii) high-performance liquid chromatography (HPLC). Furthermore, the spoilage microbiota was identified through repetitive extragenic palindrome-PCR, while selected isolates were further characterized based on sequencing of the V1-V3 region of the 16S rRNA gene. Microbial growth was observed only during storage of cream samples at 12 and 15 °C, with the applied genotypic analysis demonstrating that Bacillus subtilis subsp. subtilis was the dominant spoilage microorganism of this product. Based on the HPLC analysis results, citric acid and sucrose were the most abundant organic acid and sugar, respectively throughout storage of cream pudding, whereas notable changes mainly included: (i) increase in the concentration of lactic acid and to a lesser extent of formic and acetic acids, and (ii) increase in the concentration of glucose and fructose at the expense of sucrose and lactose. The results of this study should be useful for the dairy industry in detecting and controlling microbiological spoilage in cream pudding and other chilled, neutral-pH dairy desserts.

Evaluation of Fourier transform infrared spectroscopy and multispectral imaging as means of estimating the microbiological spoilage of farmed sea bream.

The objective of the present study was the evaluation of Fourier transform infrared (FTIR) spectroscopy and multispectral imaging (MSI), in tandem with multivariate data analysis, as means of estimating the microbiological quality of sea bream. Farmed whole ungutted fish were stored aerobically at 0, 4 and 8 °C. At regular time intervals, fish samples (i.e. cut portions) were analysed microbiologically, while FTIR and MSI measurements also were acquired at both the skin and flesh sides of the samples. Partial least squares regression (PLSR) models were calibrated to provide quantitative estimations of the microbiological status of fish based on spectral data, in a temperature-independent manner. The PLSR model based on the FTIR data of fish skin exhibited good performance when externally validated, with the coefficient of determination (R2) and the root mean square error (RMSE) being 0.727 and 0.717, respectively. Hence, FTIR spectroscopy appears to be promising for the rapid and non-invasive monitoring of the microbiological spoilage of whole sea bream. Contrarily, the MSI models' performance was unsatisfactory, delimitating their potential exploitation in whole fish quality assessment. Model optimization results concerning fish flesh indicated that MSI may be propitious in skinned fish products, with its definite competence warranting further investigation.

Online Feature Selection for Robust Classification of the Microbiological Quality of Traditional Vanilla Cream by Means of Multispectral Imaging.

The performance of an Unsupervised Online feature Selection (UOS) algorithm was investigated for the selection of training features of multispectral images acquired from a dairy product (vanilla cream) stored under isothermal conditions. The selected features were further used as input in a support vector machine (SVM) model with linear kernel for the determination of the microbiological quality of vanilla cream. Model training (n = 65) was based on two batches of cream samples provided directly by the manufacturer and stored at different isothermal conditions (4, 8, 12, and 15 °C), whereas model testing (n = 132) and validation (n = 48) were based on real life conditions by analyzing samples from different retail outlets as well as expired samples from the market. Qualitative analysis was performed for the discrimination of cream samples in two microbiological quality classes based on the values of total viable counts [TVC ≤ 2.0 log CFU/g (fresh samples) and TVC ≥ 6.0 log CFU/g (spoiled samples)]. Results exhibited good performance with an overall accuracy of classification for the two classes of 91.7% for model validation. Further on, the model was extended to include the samples in the TVC range 2-6 log CFU/g, using 1 log step to define the microbiological quality of classes in order to assess the potential of the model to estimate increasing microbial populations. Results demonstrated that high rates of correct classification could be obtained in the range of 2-5 log CFU/g, whereas the percentage of erroneous classification increased in the TVC class (5,6) that was close to the spoilage level of the product. Overall, the results of this study demonstrated that the UOS algorithm in tandem with spectral data acquired from multispectral imaging could be a promising method for real-time assessment of the microbiological quality of vanilla cream samples.

Probiotic Incorporation in Edible Films and Coatings: Bioactive Solution for Functional Foods.

Nowadays, the consumption of food products containing probiotics, has increased worldwide due to concerns regarding healthy diet and wellbeing. This trend has received a lot of attention from the food industries, aiming to produce novel probiotic foods, and from researchers, to improve the existing methodologies for probiotic delivery or to develop and investigate new possible applications. In this sense, edible films and coatings are being studied as probiotic carriers with many applications. There is a wide variety of materials with film-forming ability, possessing different characteristics and subsequently affecting the final product. This manuscript aims to provide significant information regarding probiotics and active/bioactive packaging, to review applications of probiotic edible films and coatings, and to discuss certain limitations of their use as well as the current legislation and future trends.

Variability in the adaptive acid tolerance response phenotype of Salmonella enterica strains.

The objective of this study was the assessment of the stationary-phase, low-pH-inducible acid tolerance response (ATR) of different Salmonella enterica strains. For this purpose, 30 strains of the pathogen were grown in tryptone soy broth in the absence (non-adapted cultures) and presence (1% w/v; acid-adapted cultures) of glucose, and then subjected to 4-h acid challenge trials at pH 3.0. Surviving populations of each strain were determined at 1-h intervals, and the Weibull model was fitted to the derived microbiological data. Extensive variability in the acid stress responses of the tested S. enterica strains was observed, with the total population reductions (log CFU/ml) attained in 4 h of acid challenge ranging from 0.9 to 5.5 and from 0.6 to 7.0 for the non-adapted and acid-adapted cultures, respectively. As demonstrated by the model scale parameter δ and shape parameter p, the effect of acid adaptation on the inactivation curves was strain-specific. Although acid adaptation resulted in enhanced acid survival for the majority of the tested strains, there were strains exhibiting similar or decreased acid resistance compared to their non-adapted counterparts. Moreover, acid adaptation appeared to decrease the strain variability of δ whereas increasing the strain variability of p: the coefficient of variation of δ among the tested strains was 97.2 and 54.9% for the non-adapted and acid-adapted cultures, respectively, while the corresponding values for p were 12.7 and 48.1%. The data of the present study, which is the first one to systematically evaluate the adaptive ATR of multiple S. enterica strains, clearly demonstrate that this phenotype (attempted to be induced by growing the pathogen in the presence of glucose) is strain-dependent.

Effect of different marinating conditions on the evolution of spoilage microbiota and metabolomic profile of chicken breast fillets.

Five different marinades were prepared containing lemon juice, apple cider vinegar, pomegranate juice and combinations of them. Three different temperatures (4, 10, and 20 °C) and five marinating time intervals (1, 3, 6, and 9 h) were tested. Microbial, physicochemical as well as sensory analyses were performed to assess marination. Noticeable microbial reductions and satisfactory sensory results were observed only in samples treated for short time (1 and 3 h). The marinade in which pomegranate and lemon juices were combined caused a decrease in microbial counts and led to desirable sensory attributes. Each of the marinades was characterized by a distinguishable organic acid profile, while the discrimination of the samples, based on organic acid concentration, between low (1 and 3) and high (6 and 9) marinating time was feasible. It can be concluded that marinating time affected the indigenous microbiota and the sensory characteristics of chicken meat while pomegranate could be a promising marinating ingredient from a microbiological and physicochemical perspective.

Alginate-Based Edible Films Delivering Probiotic Bacteria to Sliced Ham Pretreated with High Pressure Processing.

The aim of the present work was to evaluate the efficacy of Na-alginate edible films as vehicles for delivering probiotic bacteria to sliced ham with or without pretreatment using high pressure processing (HPP). Three strains of probiotic bacteria were incorporated in Na-alginate forming solution. Ham slices (with or without pretreatment using HPP at 500 MPa for 2 min) were packed under vacuum in contact with the films and then stored at 4, 8 and 12 °C for 66, 47 and 40 days, respectively. Microbiological analysis was performed in parallel with pH and color measurements. Sensory characteristics were assessed, while the presence and the relative abundance of each probiotic strain during storage was evaluated using pulsed field gel electrophoresis. In ham slices without HPP treatment, probiotic bacteria were enumerated above 106 CFU/g during storage at all temperatures. Same results were obtained in cases of HPP treated samples, but pH measurements showed differences with the latter ones exhibiting higher values. Sensory evaluation revealed that probiotic samples had a more acidic taste and odor than the control ones, however these characteristics were markedly compromised in samples treated with HPP. Overall, the results of the study are promising since probiotic bacteria were successfully delivered in the products by edible films regardless of the HPP treatment.

Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms.

UNLABELLED: Salmonella is recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spice Thymbra capitata against both planktonic and biofilm cells of Salmonella enterica serovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined by in situ and real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure of Salmonella biofilm. IMPORTANCE: The results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol of Thymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

Development of a predictive model for the growth kinetics of aerobic microbial population on pomegranate marinated chicken breast fillets under isothermal and dynamic temperature conditions.

The aim of this study was the development of a model to describe the growth kinetics of aerobic microbial population of chicken breast fillets marinated in pomegranate juice under isothermal and dynamic temperature conditions. Moreover, the effect of pomegranate juice on the extension of the shelf life of the product was investigated. Samples (10 g) of chicken breast fillets were immersed in marinades containing pomegranate juice for 3 h at 4 °C following storage under aerobic conditions at 4, 10, and 15 °C for 10 days. Total Viable Counts (TVC), Pseudomonas spp and lactic acid bacteria (LAB) were enumerated, in parallel with sensory assessment (odor and overall appearance) of marinated and non-marinated samples. The Baranyi model was fitted to the growth data of TVC to calculate the maximum specific growth rate (µmax) that was further modeled as a function of temperature using a square root-type model. The validation of the model was conducted under dynamic temperature conditions based on two fluctuating temperature scenarios with periodic changes from 6 to 13 °C. The shelf life was determined both mathematically and with sensory assessment and its temperature dependence was modeled by an Arrhenius type equation. Results showed that the µmax of TVC of marinated samples was significantly lower compared to control samples regardless temperature, while under dynamic temperature conditions the model satisfactorily predicted the growth of TVC in both control and marinated samples. The shelf-life of marinated samples was significantly extended compared to the control (5 days extension at 4 °C). The calculated activation energies (Ea), 82 and 52 kJ/mol for control and marinated samples, respectively, indicated higher temperature dependence of the shelf life of control samples compared to marinated ones. The present results indicated that pomegranate juice could be used as an alternative ingredient in marinades to prolong the shelf life of chicken.

Biofilm formation on Conservolea natural black olives during single and combined inoculation with a functional Lactobacillus pentosus starter culture.

The potential of biofilm formation of multifunctional starters Lactobacillus pentosus B281 and Pichia membranifaciens M3A during inoculated fermentation of Conservolea natural black olives according to Greek-style processing was investigated. Olives were directly brined in 8% (w/v) NaCl following three fermentation procedures namely, i) spontaneous fermentation, ii) inoculated fermentation with L. pentosus B281, and iii) co-inoculated fermentation with L. pentosus B281 and P. membranifaciens M3A. Lactic acid bacteria (LAB) and yeasts were monitored on olives by plate counting for a period of 153 days, whereas the survival of the inoculated strains was confirmed by Pulsed Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism (RFLP) analysis. Inoculated fermentation with L. pentosus B281 with/without the presence of the yeast resulted in higher acidification of the brine compared to the spontaneous process where no indigenous LAB could be enumerated. The population of LAB on olives ranged between 5.5 and 6.5 log CFU/g and it was maintained at higher levels compared to yeasts (3.5-4.5 log CFU/g) throughout the process. PFGE analysis revealed that L. pentosus B281 could successfully colonize the surface of black olives presenting high recovery rate (100%) at the end of fermentation in contrast to P. membranifaciens M3A that was successfully recovered (42%) only after 72 days of the process. The obtained results provide interesting perspectives for the production of natural black olives with functional properties.

Microbiological spoilage and volatiles production of gutted European sea bass stored under air and commercial modified atmosphere package at 2 °C.

Microbiological, sensory, TVB-N and TMA-N changes and Volatile Organic Compounds (VOCs) detection using the SPME/GC-MS technique, were performed to evaluate potential chemical spoilage indices (CSI) of gutted sea bass (Dicentrarchus labrax) stored at 2 °C under air and in modified atmosphere packaging (MAP CO2: 60%, O2: 10%, N2: 30%). Shelf-life, determined by sensory evaluation, of gutted sea bass stored at 2 °C under air and MAP was 9 and 13 d respectively. Pseudomonas and H2S producing bacteria were among the dominant spoilage microorganisms under both storage conditions, while Lactic Acid Bacteria (LAB) and Brochothrix thermosphacta were co-dominant with Pseudomonas and H2S producing bacteria under MAP. The traditional CSIs such as TVB-N and TMA-N were increased substantially only at the late stages of storage or after rejection of the products, making them unsuitable for freshness/spoilage monitoring throughout storage. A substantial number of VOCs attributed to microbiological action or chemical activity, were detected including alcohols, aldehydes, ketones, organic acids and esters. The level of microbial origin VOCs such as ethanol, 2-ethyl-1-hexanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methylbutanal, 2-methylbutanal and some ethyl esters increased during storage, suggesting their potential as CSIs.

Quantification of Aspergillus carbonarius in grapes using a real time PCR assay.

A study on the representation of Aspergillus carbonarius in the vineyards of the Mesogeia geographical region of Attica, Greece, was conducted. One hundred and twenty five samples of the indigenous drought and disease resistant Savatiano wine grape variety, the most widely planted in Greece, were collected. The sample's total DNA extracts were initially tested for fungal DNA presence by targeting the Internal Transcribed Spacer (ITS) region in end-point Polymerase Chain Reactions. Samples which were proved positive were further subjected to PCR analysis using specific primers targeting an A. carbonarius polycetide synthase (pks) gene. Among ITS positive samples (70%), A. carbonarius was represented in 42% of them. Furthermore, a SYBR Green I Real Time PCR method was used to quantify the amount of this species in the grape samples. The values of the positive samples were estimated in the range of 13 to 50 × 10(3) fungal haploid genomes/g grapes. The significance of this study lies in the applicability of a rapid and culture-independent method to detect and quantify A. carbonarius on grapes.

Bacterial populations and the volatilome associated to meat spoilage.

Microbial spoilage of meat is a complex event to which many different bacterial populations can contribute depending on the temperature of storage and packaging conditions. The spoilage can derive from microbial development and consumption of meat nutrients by bacteria with a consequent release of undesired metabolites. The volatile organic compounds (VOCs) that are generated during meat storage can have an olfactory impact and can lead to rejection of the product when their concentration increase significantly as a result of microbial development. The VOCs most commonly identified in meat during storage include alcohols, aldehydes, ketones, fatty acids, esters and sulfur compounds. In this review, the VOCs found in fresh meat during storage in specific conditions are described together with the possible bacterial populations responsible of their production. In addition, on the basis of the data available in the literature, the sensory impact of the VOCs and their dynamics during storage is discussed to highlight their possible contribution to the spoilage of meat.

Modelling the influence of temperature, water activity and sodium metabisulphite on the growth and OTA production of Aspergillus carbonarius isolated from Greek wine grapes.

The purpose of the present study was to develop a modelling approach to quantify the effect of temperature (15-38 °C), a(w) (0.88-0.98) and sodium metabisulphite (NaMBS) concentration (0-200 mg L(-1)) on the growth and OTA production of Aspergillus carbonarius on a Grape Juice based Medium (GJM). Growth responses of the fungus were recorded over time in terms of colony diameter changes, and fitted to the primary model of Baranyi and the estimated maximum growth rates (µ(max)) and lag phases (λ) were subsequently modelled as a function of temperature, a(w) and NaMBS concentration using the cardinal values model with inflection (CMI). Moreover, OTA production was measured during fungal growth and modelled as a function of the same parameters through a quadratic polynomial model. Results showed that NaMBS increased the lag phase of A. carbonarius, particularly at 38 °C/0.98 a(w) and 38 °C/0.96 a(w), as well as at lower a(w) levels regardless of temperature. In the lowest NaMBS concentration (50 mg L(-1)) there was no inhibitory effect, while at higher concentrations (100 and 150 mg L(-1)) fungal growth was delayed. No growth was observed at 200 mg L(-1) of NaMBS irrespective of temperature and a(w) levels. The optimum values for growth were found in the range 30-35 °C and 0.96 a(w), while for OTA production at 20 °C and 0.98 a(w). The developed models were subjected to internal and external validation and presented satisfactory performance as inferred by graphical plots and statistical indices (bias and accuracy factors). The present study will complement the findings on the ecophysiology of A. carbonarius using NaMBS as an inhibitory agent.

Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives.

The performance of two strains of lactic acid bacteria (LAB), namely Lactobacillus pentosus B281 and Lactobacillus plantarum B282, previously isolated from industrially fermented table olives and screened in vitro for probiotic potential, was investigated as starter cultures in Spanish style fermentation of cv. Halkidiki green olives. Fermentation was undertaken at room temperature in two different initial salt concentrations (8% and 10%, w/v, NaCl) in the brines. The strains were inoculated as single and combined cultures and the dynamics of their population on the surface of olives was monitored for a period of 114 days. The survival of inoculated strains on olives was determined using Pulsed Field Gel Electrophoresis (PFGE). Both probiotic strains successfully colonized the olive surface at populations ranged from 6.0 to 7.0 log CFU/g throughout fermentation. PFGE analysis revealed that L. pentosus B281 presented higher colonization in both salt levels at the end of fermentation (81.2% and 93.3% in 8% and 10% NaCl brines, respectively). For L. plantarum B282 a high survival rate (83.3%) was observed in 8% NaCl brines, but in 10% NaCl the strain could not colonize the surface of olives. L. pentosus B281 also dominated over L. plantarum B282 in inoculated fermentations when the two strains were used as combined culture. The biochemical profile (pH, organic acids, volatile compounds) attained during fermentation and the sensory analysis of the final product indicated a typical lactic acid fermentation process of green olives.