Delving into the study of lactic acid bacteria and yeasts distribution in table olive biofilms using a non-destructive procedure.

To turn table olives into appropriate carriers of beneficial bacteria and yeasts to consumers, it is essential to have reliable methods for analysing microorganisms in biofilms. This work validates the application of a non-destructive procedure to study the lactic acid bacteria and yeasts distribution in fruits during Spanish-style green table olive fermentations. Laboratory-scale fermentations were inoculated simultaneously with three Lactiplantibacillus pentosus strains (LPG1, 119, and 13B4) and two yeasts (Wickerhamomyces anomalus Y12 and Saccharomyces cerevisiae Y30), all of them natives of table olive fermentations. Data showed that L. pentosus LPG1 and yeasts W. anomalus Y12 were quite prone to colonise olive biofilms, but only the Lactiplantibacillus strain also can penetrate the epidermis of the fruit and colonise the flesh. Applying a non-destructive treatment consisting in shelling the fruits with glass beads led to obtaining similar lactic acid bacteria and yeast recovery than the classical stomacher destructive method. However, the glass bead procedure improved the quality of the metagenomics analysis (especially when using 16 S rRNA gene-based sequencing). Results show the great utility of procedures that do not destroy the fruit for studying fermented vegetable biofilms.

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.

Relating starter cultures to volatile profile and potential markers in green Spanish-style table olives by compositional data analysis.

This work relates native lactic acid bacteria (LAB) (Lactobacillus pentosus LPG1, L. pentosus Lp13, and Lactobacillus plantarum Lpl15) and yeast (Wickerhamomyces anomalus Y12) starters to the volatile components (VOCs) produced in green Spanish-style table olives. For this aim, the VOC profile was considered as compositional data (CoDa). The CoDa analysis generated new information on the relationship among inocula and VOCs through the tetrahedral plot, CoDa-biplot, variation array matrix, and CoDa dendrogram. The ilr (which includes pivot) coordinates (Euclidean space) from VOCs produced more reliable starters' clustering than the original data. The potential VOC markers, identified by a test based on the pairwise comparison of the logratio variation arrays from the whole data set and the individual groups, were (starters in the parenthesis): 2-phenylethyl acetate (LPG1, Y12, Y12 + LAB), methanol (Lpl15), cis-2-penten-1-ol (LPG1, Y12, Y12 + LAB), 2-methyl-3-hexanol (LPG1, Y12), U (non-identified) C (m/z 83-112-97) (Y12) and UF (m/z 95-154-110) (LPG1, Y12 + LAB). Besides, some VOCs were partial/totally inhibited by specific starters: 2-methyl-1-propanol (Lp13, Y12 + LAB), 2-phenyl ethanol (Lp13), furfuryl methyl ether (Y12 + LAB), purpurocatechol (Y12, Y12 + LAB), 4-ethyl guaiacol (Lp13, Lpl15), 4-ethyl phenol (Lpl15), 5-tert-butylpyrogallol (Lp13, Lpl15), and UE (m/z 111-198) (Lp13). A better understanding of the relationship between starters and their VOC may facilitate modelling the flavour and quality of Spanish-style green table olive fermentations.

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.

Genotyping, identification and multifunctional features of yeasts associated to Bosana naturally black table olive fermentations.

Directly brined black table olives of Bosana variety are a traditional food product of Sardinia island (Italy), spontaneously fermented by yeasts among other microorganisms. However, as far as we know, the identification, biotechnological and probiotic potential of this yeast community has not been investigated yet. In this work, a total of 72 yeast isolates previously obtained from Bosana olive brines were first genotyped by Random Amplified Polymorphic DNA (RAPD-PCR) analysis with primer M13, and then identified by sequencing of D1/D2 domains of rDNA 26S gene. The dominant species were Wickerhamomyces anomalus and Nakazawaea molendini-olei, albeit Candida diddensiae, Candida boidinii, Zygotorulaspora mrakii, and Saccharomyces cerevisiae were also present in lower proportions. For the different biotypes of yeasts obtained, the multivariate analysis of their technological (esterase, lipase and ß-glucosidase activities, growth in presence of oleuropein, resistance and susceptibility to NaCl) and probiotic (removal of cholesterol, gastric and pancreatic digestions, biofilms assays alone and in co-culture with Lactobacillus pentosus) features, showed that W. anomalus Wa1 exhibited the best technological characteristics, while S. cerevisiae Sc24 and C. boidinii Cb60 showed promising probiotic features. Therefore, they may have potential application as multifunctional starters, alone or in combination with lactic acid bacteria, during olive processing, albeit further studies are necessary to validate these results.

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.

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.

Main Challenges Expected from the Impact of Climate Change on Microbial Biodiversity of Table Olives: Current Status and Trends.

Climate change is a global emergency that is affecting agriculture in Mediterranean countries, notably the production and the characteristics of the final products. This is the case of olive cultivars, a source of olive oil and table olives. Table olives are the most important fermented vegetables in the Mediterranean area, whose world production exceeds 3 million tons/year. Lactic acid bacteria and yeast are the main microorganisms responsible for the fermentation of this product. The microbial diversity and population dynamics during the fermentation process are influenced by several factors, such as the content of sugars and phenols, all of which together influence the quality and safety of the table olives. The composition of fruits is in turn influenced by environmental conditions, such as rainfall, temperature, radiation, and the concentration of minerals in the soil, among others. In this review, we discuss the effect of climate change on the microbial diversity of table olives, with special emphasis on Spanish and Portuguese cultivars. The alterations expected to occur in climate change scenario(s) include changes in the microbial populations, their succession, diversity, and growth kinetics, which may impact the safety and quality of the table olives. Mitigation and adaptation measures are proposed to safeguard the authenticity and sensorial features of this valuable fermented food while ensuring food safety requirements.

Understanding the transcriptomic response of Lactiplantibacillus pentosus LPG1 during Spanish-style green table olive fermentations.

Lactiplantibacillus pentosus (Lbp. pentosus) is a species of lactic acid bacteria with a great relevance during the table olive fermentation process, with ability to form non-pathogenic biofilms on olive epidermis. The objective of this work is to deepen into the genetic mechanisms of adaptation of Lpb. pentosus LPG1 during Spanish-style green table olive fermentations, as well as to obtain a better understanding of the mechanisms of adherence of this species to the fruit surface. For this purpose, we have carried out a transcriptomic analysis of the differential gene expression of this bacterium during 60 days of fermentation in both brine and biofilms ecosystems. In brines, it was noticed that a total of 235 genes from Lpb. pentosus LPG1 were differentially expressed during course of fermentation and grouped into 9 clusters according to time-course analysis. Transport and metabolism of carbohydrates and amino acids, energy production, lactic acid and exopolysaccharide synthesis genes increased their expression in the planktonic cells during course of fermentation. On the other hand, expression of genes associated to stress response, bacteriocin synthesis and membrane protein decreased. A total of 127 genes showed significant differential expression between Lpb. pentosus LPG1 planktonic (brine) and sessile (biofilms) cells at the end of fermentation process (60 days). Among the 64 upregulated genes in biofilms, we found genes involved in adhesion (strA), exopolysaccharide production (ywqD, ywqE, and wbnH), cell shape and elongation (MreB), and well as prophage excision. Deeping into the genetic bases of beneficial biofilm formation by Lpb. pentosus strains with probiotic potential will help to turn this fermented vegetable into a carrier of beneficial microorganisms to the final consumers.

In Silico Evidence of the Multifunctional Features of Lactiplantibacillus pentosus LPG1, a Natural Fermenting Agent Isolated from Table Olive Biofilms.

In recent years, there has been a growing interest in obtaining probiotic bacteria from plant origins. This is the case of Lactiplantibacillus pentosus LPG1, a lactic acid bacterial strain isolated from table olive biofilms with proven multifunctional features. In this work, we have sequenced and closed the complete genome of L. pentosus LPG1 using both Illumina and PacBio technologies. Our intention is to carry out a comprehensive bioinformatics analysis and whole-genome annotation for a further complete evaluation of the safety and functionality of this microorganism. The chromosomic genome had a size of 3,619,252 bp, with a GC (Guanine-Citosine) content of 46.34%. L. pentosus LPG1 also had two plasmids, designated as pl1LPG1 and pl2LPG1, with lengths of 72,578 and 8713 bp (base pair), respectively. Genome annotation revealed that the sequenced genome consisted of 3345 coding genes and 89 non-coding sequences (73 tRNA and 16 rRNA genes). Taxonomy was confirmed by Average Nucleotide Identity analysis, which grouped L. pentosus LPG1 with other sequenced L. pentosus genomes. Moreover, the pan-genome analysis showed that L. pentosus LPG1 was closely related to the L. pentosus strains IG8, IG9, IG11, and IG12, all of which were isolated from table olive biofilms. Resistome analysis reported the absence of antibiotic resistance genes, whilst PathogenFinder tool classified the strain as a non-human pathogen. Finally, in silico analysis of L. pentosus LPG1 showed that many of its previously reported technological and probiotic phenotypes corresponded with the presence of functional genes. In light of these results, we can conclude that L. pentosus LPG1 is a safe microorganism and a potential human probiotic with a plant origin and application as a starter culture for vegetable fermentations.

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.

Application of Compositional Data Analysis to Study the Relationship between Bacterial Diversity in Human Faeces and Sex, Age, and Weight.

This work uses Compositional Data Analysis (CoDA) to examine the typical human faecal bacterial diversity in 39 healthy volunteers from the Andalusian region (Spain). Stool samples were subjected to high-throughput sequencing of the V3 and V4 regions of the 16S ribosomal RNA gene using Illumina MiSeq. The numbers of sequences per sample and their genus-level assignment were carried out using the Phyloseq R package. The alpha diversity indices of the faecal bacterial population were not influenced by the volunteer's sex (male or female), age (19-46 years), and weight (48.6-99.0 kg). To study the relationship between these variables and the faecal bacterial population, the ALDEx2 and coda4microbiome CoDA packages were used. Applying ALDEx2, a trend suggesting a connection between sex and the genera Senegalimassilia and Negatibacillus (slightly more abundant in females) and Desulfovibrio (more abundant in males) was found. Moreover, age was tentatively associated with Streptococcus, Tizzerella, and Ruminococaceae_UCG-003, while weight was linked to Senegalimassilia. The exploratory tool of the coda4microbiome package revealed numerous bacterial log-ratios strongly related to sex and, to a lesser extent, age and weight. Moreover, the cross-sectional analysis identified bacterial signature balances able to assign sex to samples regardless of controlling for volunteers' age or weight. Desulfovibrio, Faecalitalea, and Romboutsia were relevant in the numerator, while Coprococcus, Streptococcus, and Negatibacillus were prominent in the denominator; the greater presence of these could characterise the female sex. Predictions for age included Caproiciproducens, Coprobacter, and Ruminoclostridium in the numerator and Odoribacter, Ezakiella, and Tyzzerella in the denominator. The predictions depend on the relationship between both groups, but the abundance of the first group and scarcity of the second could be related to older individuals. However, the association of the faecal bacterial population with weight did not yield a satisfactory model, indicating scarce influence. These results demonstrate the usefulness of the CoDA methodology for studying metagenomics data and, specifically, human microbiota.

Modeling the antimicrobial effects of olive mill waste extract, rich in hydroxytyrosol, on the growth of lactic acid bacteria using response surface methodology.

The objective of this study is to assess the inhibitory effects of an aqueous extract from olive oil mill waste (alperujo) on the growth of a lactic acid bacteria (LAB) cocktail consisting of various strains of Lactiplantibacillus pentosus and Lactiplantibacillus plantarum species. For this purpose, response surface methodology was employed using two independent variables (pH levels 3.5-5.55; hydroxytyrosol concentration ranging from 0.93-2990 ppm). The response variable was the average inhibition per treatment on the LAB cocktail (expressed as a percentage). The developed model identified significant terms, including the linear effect of hydroxytyrosol and pH, their interaction, and the quadratic effect of pH. Maximum inhibition of the LAB cocktail was observed at progressively higher concentrations of hydroxytyrosol and lower pH values. Therefore, complete inhibition of LAB in the synthetic culture medium could only be achieved for concentrations of 2984 ppm hydroxytyrosol at a pH of 3.95. These findings suggest that extracts derived from "alperujo" could be utilized as a natural preservative in acidified foods with a bitter flavor and antioxidant requirements.

Oral intake of Lactiplantibacillus pentosus LPG1 Produces a Beneficial Regulation of Gut Microbiota in Healthy Persons: A Randomised, Placebo-Controlled, Single-Blind Trial.

The search for vegetable-origin probiotic microorganisms is a recent area of interest. This study conducted a phase I clinical trial to assess the effects of oral administration of Lactiplantibacillus pentosus LPG1, a natural strain with probiotic potential isolated from table olive fermentations, on the gut microbiota. The trial was a randomised, placebo-controlled, single-blind study involving 39 healthy volunteers. Group A (n = 20) ingested one capsule/day of L. pentosus LPG1 containing 1 × 1010 UFC/capsule, while Group B (n = 19) received one capsule/day containing only dextrose (placebo). The capsules were taken during breakfast for 30 consecutive days. Human stool samples were collected from all volunteers at the beginning (baseline) and at the end of the study (post-intervention) and were subjected to 16S rRNA metataxonomic analysis using Illumina MiSeq. Sequencing data at the genus level were statistically analysed using traditional methods and compositional data analysis (CoDA). After treatment, the alpha diversity in Group B (placebo) decreased according to an increase in the Berger and Parker dominance index (p-value < 0.05); moreover, dominance D increased and Simpson 1-D index decreased (p-value < 0.10). The Lactobacillus genus in the faeces was included in the CoDA signature balances (selbal and coda4microbiome) and played a notable role in distinguishing samples from baseline and post-intervention in Group A (LPG1). Additionally, ingesting L. pentosus LPG1 modified the gut microbiota post-intervention, increasing the presence of Parabacteroides and Agathobacter, but reducing Prevotella. These findings suggest that L. pentosus LPG1 is a potentially beneficial gut microbiota modulator in healthy persons.

Strategies for the Identification and Assessment of Bacterial Strains with Specific Probiotic Traits.

Early in the 1900s, it was proposed that health could be improved and senility delayed by manipulating gut microbiota with the host-friendly bacteria found in yogurt. Later, in 1990, the medical community reconsidered this idea and today probiotics represent a developed area of research with a billion-dollar global industry. As a result, in recent decades, increased attention has been paid to the isolation and characterization of novel probiotic bacteria from fermented foods and dairy products. Most of the identified probiotic strains belong to the lactic acid bacteria group and the genus Bifidobacterium. However, current molecular-based knowledge has allowed the identification and culture of obligatory anaerobic commensal bacteria from the human gut, such as Akkermansia spp. and Faecalibacterium spp., among other human symbionts. We are aware that the identification of new strains of these species does not guarantee their probiotic effects and that each effect must be proved through in vitro and in vivo preclinical studies before clinical trials (before even considering it as a probiotic strain). In most cases, the identification and characterization of new probiotic strain candidates may lack the appropriate set of in vitro experiments allowing the next assessment steps. Here, we address some innovative strategies reported in the literature as alternatives to classical characterization: (i) identification of alternatives using whole-metagenome shotgun sequencing, metabolomics, and multi-omics analysis; and (ii) probiotic characterization based on molecular effectors and/or traits to target specific diseases (i.e., inflammatory bowel diseases, colorectal cancer, allergies, among others).

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.

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.

Behavior of Vibrio spp. in Table Olives.

The presence of Vibrio species in table olive fermentations has been confirmed by molecular biology techniques in recent studies. However, there has been no report of any foodborne outbreak caused by Vibrio due to the consumption of table olives, and their role as well as the environmental conditions allowing their survival in table olives has not been elucidated so far. The aims of this work were to model the behavior of an inoculated Vibrio cocktail in diverse table olive environments and study the possible behavior of an inoculated Vibrio cocktail in table olives. First, an in vitro study has been performed where the microbial behavior of a Vibrio cocktail was evaluated in a laboratory medium and in olive brines using predictive models at different NaCl concentrations (2-12%) and pH levels (4.0-9.0). Afterward, a challenge testing was done in lye-treated olives inoculated at the beginning of fermentation with the Vibrio cocktail for 22 days. The Vibrio cocktail inoculated in table olives has not been detected in olive brines during fermentation at different pH levels. However, it was observed that this microorganism in a laboratory medium could reach an optimal growth at pH 9 and 2% salt, without time of constant absorbance (t A), and the maximum absorbance value (y end) observed was at pH 8 and 2% salt conditions. The statistical analysis demonstrated that the effect of salt concentration was higher than pH for the kinetic growth parameters (µmax, t A, and y end). On the other hand, it was confirmed that no growth of the Vibrio cocktail on any sample was noticed in lye-treated olive fermentations. Thus, it was concluded that the presence of olive compounds (unknown) did not allow the development of Vibrio strains, so it is a very safety product as it has a natural antimicrobial compound, but the possibility that a native Vibrio sp. is able to acquire the capacity to adapt to this compound should be considered in further studies.

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.