Probiotic and antifungal attributes of lactic acid bacteria isolates from naturally fermented Brazilian table olives

Citation: Simões, L.; Fernandes, N.; de Souza, A.; dos Santos, L.; Magnani, M.; Abrunhosa, L.; Teixeira, J.; Schwan, R.F.; Dias, D.R. Probiotic and Antifungal Attributes of Lactic Acid Bacteria Isolates from Naturally Fermented Brazilian Table Olives. Fermentation 2022,8, 277. https:// doi.org/10.3390/fermentation8060277 Academic Editor: Amparo Gamero Received: 25 May 2022 Accepted: 11 June 2022 Published: 14 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). fermentation Article Probiotic and Antifungal Attributes of Lactic Acid Bacteria Isolates from Naturally Fermented Brazilian Table Olives Luara Simões 1,2 , Natália Fernandes 1,3 , Angélica de Souza 1, Luiz dos Santos 4, Marciane Magnani 5, Luís Abrunhosa 6,7 , JoséTeixeira 6,7 , Rosane Freitas Schwan 1,* and Disney Ribeiro Dias 8,* 1 Biology Department, Federal University of Lavras, Lavras 36200-900, Brazil; [email protected] (L.S.); [email protected] (N.F.); [email protected] (A.d.S.) 2Centre of Molecular and Environmental Biology, University of Minho, 4710-057 Braga, Portugal 3Chemistry Department, University of California, Davis, CA 95616, USA 4Nutrition Department, Federal University of Lavras, Lavras 36200-900, Brazil; [email protected] 5Food Engineering Department, Federal University of Paraíba, Paraíba 58051-900, Brazil; [email protected] 6CEB—Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; [email protected] (L.A.); [email protected] (J.T.) 7LABBELS—Associate Laboratory, 4800-122 Guimarães, Portugal 8Department of Food Science, Federal University of Lavras, Lavras 36200-900, Brazil *Correspondence: [email protected] (R.F.S.); [email protected] (D.R.D.); Tel.: +55-(35)-3829-1614 (R.F.S.); +55-(35)-3829-5256 (D.R.D.) Abstract: Research with fermented olives as a source of wild Lactic Acid Bacteria (LAB) strains with probiotic and biotechnological characteristics constitutes a promising field of work. The present study evaluated in vitro probiotic, antifungal, and antimycotoxigenic potential of LAB isolates from naturally fermented Brazilian table olives. Among fourteen LAB isolates, the Levilactobacillus brevis CCMA 1762, Lactiplantibacillus pentosus CCMA 1768, and Lacticaseibacillus paracasei subsp. paracasei CCMA 1770 showed potential probiotic and antifungal properties. The isolates showed resistance to pH 2.0 (survival ≥ 84.55), bile salts (survival ≥ 99.44), and gastrointestinal tract conditions (survival ≥ 57.84%); hydrophobic cell surface ( ≥ 27%); auto-aggregation ( ≥ 81.38%); coaggregation with Escherichia coli INCQS 00181 ( ≥ 33.97%) and Salmonella Enteritidis ATCC 564 ( ≥ 53.84%); adhesion to the epithelial cell line Caco-2 ( ≥ 5.04%); antimicrobial activity against the bacteria S. Enteritidis ATCC 564 ( ≥ 6 mm), Listeria monocytogenes ATCC 19117 ( ≥ 6 mm), Staphylococcus aureus ATCC 8702 ( ≥ 3 mm), and the fungi Penicillium nordicum MUM 08.16 (inhibition ≥ 64.8%). In addition, the strains showed the ability to adsorb the mycotoxins aflatoxin B1 ( ≥ 40%) and ochratoxin A ( ≥ 34%). These results indicate that LAB strains from naturally fermented Brazilian table olives are potentially probiotic and antifungal candidates that can be used for food biopreservation. Keywords: adhesion ability; antifungal activity; fermented olives; in vitro digestive simulation; mycotoxins; probiotic selection 1. Introduction In addition to benefiting human health, table olives are greatly appreciated worldwide for their nutritional value and sensory characteristics due to phenols and antioxidant compounds in their composition [ 1 ]. However, olive drupes cannot be eaten immediately after harvest due to oleuropein (which is responsible for the bitterness). Therefore, this fruit needs to go through several processing methods to become edible, including methods involving chemical treatment of fruits (Sevillian and Californian methods) and the natural method of fermentation (olives processed in brine without pretreatment with chemicals) [ 2 ]. The consumption of table olives is widespread throughout the world. In the 2020–2021 season, the production of table olives exceeded 2.6 million tons, with Spain and Greece Fermentation 2022,8, 277. https://doi.org/10.3390/fermentation8060277 https://www.mdpi.com/journal/fermentation Fermentation 2022,8, 277 2 of 18 being the main contributors in the Mediterranean [ 3 , 4 ]. Although olives are widely consumed in Brazil, their production is low, and most of them are imported (approximately 10,367.9 tons of table olives until August 2021) [ 3 ]. Therefore, studies on Brazilian olives are important for the characterization and valorization of this product. Table olives can contain microorganisms with biotechnological properties to be explored as a fermented food item. The natural fermentation process of olives occurs in extreme environments (olive and brine), characterized by low nutrients and high levels of polyphenols and sodium chloride. The microbiota surviving the fermentation process mainly comprises lactic acid bacteria (LAB) and yeasts. The dominant microorganisms in this environment have important physiological properties that allow their use as probiotics [5]. Therefore, research with fermented olives as a source of wild LAB strains with probiotic and technological characteristics constitutes a promising field of work [ 6 ]. Many recent works have demonstrated several health benefits of probiotic LAB found in fermented olives. The most studied activities were mainly antibacterial, antifungal, anti-adherent, antiproliferative, anticancer, metal detox, and immunostimulant [ 7 – 9 ]. However, there are no data regarding LAB isolated from fermented Brazilian table olives about probiotic potential or antifungal activity. The criteria for selecting probiotic LAB strains are safety, including the absence of the secretion of harmful metabolites, survival in gastrointestinal conditions, and the ability to inhibit pathogens [ 10 , 11 ]. The most studied probiotic properties include resistance to low pH and bile salts or tolerance toward gastric juice and pancreatic digestion—in some cases, all of them—with the main objective of achieving the survival of probiotics in the gastrointestinal tract (GIT) [ 12 ]. Recommended safety criteria in selecting probiotics include isolation environment, taxonomic identification, and absence of virulence and transferable antibiotic resistance genes [13]. The high acidification capacity of LAB is necessary to guarantee the biotransformation of the olives and the inhibiting pathogenic microorganisms responsible for deterioration [ 14 ]. Due to this important capacity, LAB has been intensively studied as a bioprotective agent and can be used to prevent fungal contamination and mycotoxin production [ 15 ]. So, using lactic acid bacteria with probiotic potential and biopreservation capacity confirms that LAB can be biological agents with high and promising applicability in food safety [ 16 ]. Although many probiotic LAB strains are characterized, the search for new probiotic strains continues to be of interest due to the vast possibilities of using these microorganisms, especially their incorporation into food matrices [ 17 ]. However, given the lack of knowledge in this sector of olives in Brazil, this study evaluated the in vitro probiotic potential, antifungal activities, and adsorption of mycotoxins of LAB isolates from naturally fermented Brazilian table olives. 2. Materials and Methods 2.1. Microorganisms and Growth Conditions A total of fourteen LABs were studied to assess the probiotic potential of each. The species Levilactobacillus brevis (L. brevis), Lacticaseibacillus paracasei subsp. paracasei (L. paracasei), and Lactiplantibacillus pentosus (L. pentosus) (Table 1) were evaluated. The strains were isolated from naturally fermented Brazilian table olives (Greek-style) of two cultivars, Grappolo 541 and Ascolano, harvested at the green stage from the Experimental Farm of EPAMIG (Minas Gerais Agricultural Research Company) in the city of Maria da Fé-MG, Brazil (22◦180south latitude and 45◦230west longitude). Fermentation 2022,8, 277 3 of 18 Table 1. Bacteria strains from table olives used in this study (n= 14). Bacteria Strain N◦of Isolates Code Cultivar and Fermentation Time Levilactobacillus brevis 3 CCMA1762 Ascolano (time 120 days) CCMA1765 Ascolano (fruit) CCMA1766 Ascolano (time 60 days) Lacticaseibacillus paracasei subsp. paracasei 10 CCMA1763 Ascolano (time 120 days) CCMA1764 Ascolano (time 60 days) CCMA1767 Ascolano (time 30 days) CCMA1769 Ascolano (time 120 days) CCMA1770 Grappolo (time 60 days) CCMA1771 Grappolo (fruit) CCMA1772 Ascolano (time 60 days) CCMA1773 Ascolano (fruit) CCMA1774 Grappolo (time 120 days) CCMA1775 Grappolo (time 30 days) Lactiplantibacillus pentosus 1CCMA1768 Ascolano (time 120 days) Lacticaseibacillus paracasei subsp. paracasei 1 LBC-81 (Danisco A/S, Copenhagen, Denmark) Strains showing potential properties for use as probiotics during the simulated digestion are bolded. The identification of strains was performed by MALDI-TOF, using the Maldi-tof Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany), and the cluster was identified through sequence analysis of the 16S rRNA gene using primers 27-F (5 0 -AGAGTT TGATCCTGGCTCAG-3 0 ) and 1512-R (5 0 -GGCTACCTTGTTACGACT-3 0 ) (Simões et al., 2021). All isolates were deposited in the Culture Collection of Agricultural Microbiology (CCMA) of the Federal University of Lavras. CCMA is registered with the World Data Centre for Microorganisms (WDCM) as CCMA-UFLA under WDCM 1083. For their use, each LAB strain was cultivated in Man Rogosa and Sharpe (MRS) (Oxoid, Basingstoke, UK) broth and incubated at 37 ◦C for 48 h. The bacteria pathogenic strains employed in the coaggregation and antibacterial activity assays were Escherichia coli (EPEC) INCQS 00181 CDC 055, Salmonella Enteritidis ATCC 564, Listeria monocytogenes ATCC 19117, and Staphylococcus aureus ATCC 8702. The pathogens were grown at 37 ◦ C for 24 h in BHI (Brain heart infusion, Himedia, Einhausen, Germany). The reference strain selected for all assays was the lyophilized Lacticaseibacillus paracasei subsp. paracasei LBC-81 (Danisco A/S, Copenhagen, Denmark) bacterium (Fonseca, et al. [ 18 ]. This microorganism was reactivated in MRS broth at 37 ◦ C for 48 h. A growth curve was elaborated to standardize LAB, pathogens, and the reference strain inoculum; absorbance was measured at 600 nm, and colony counting was performed using an MRS agar for LAB and a BHI agar for pathogens. The plates were incubated at 37 ◦ C for 48 h, and the inoculum was standardized at 7 to 8 log CFU mL−1. The fungal strains used in antifungal activity were Aspergillus flavus MUM 08.201 and Penicillium nordicum MUM 08.16, obtained from the MUM Culture Collection (Micoteca da Universidade do Minho, Braga, Portugal). The fungi were reactivated on MEA agar (Malt Extract Agar, Himedia, Einhausen, Germany) at 25 ◦ C in the dark. After 7 days, the fungal spores were suspended in a solution of 0.1% peptone and 0.05% Tween 80 (Fisher Scientific, Waltham, MA, USA). The concentration was standardized at 10 6 spores/mL using a Neubauer chamber. 2.2. Tolerance to Low pH and Bile Salts The fourteen LAB isolates were tested for tolerance to acid pH and bile salt concentration, according to Ramos et al. [ 19 ], with certain modifications. The isolates were grown and centrifuged (3000 × gfor 5 min at 4 ◦ C), after which the strains were resuspended in MRS broth with pH adjusted to 2.0 using an acid solution (1 N HCl) or supplemented with Fermentation 2022,8, 277 4 of 18 bile salts (Oxgall, Neogen, Lansing, MI, USA) at 0.3 % (v/v). The cells were incubated for 3 h at 37 ◦ C. Samples were obtained at the beginning (time 0 h) and end of incubation (time 3 h) to determine the total viable count. Dilutions were made (up to 10 7 ) and spread plated onto MRS agar incubated at 37 ◦ C for 48 h; viable cells were counted (log CFU mL −1 ). The percentage of survival at pH 2.0 or bile salt was calculated using the equation: Survival at pH 2 (%) or bile salt (%) = Final viable count/Initial viable count ×100 2.3. Cell Surface Hydrophobicity For twelve strains selected in the previous assay, the cell surface hydrophobicity assay was performed according to dos Santos, et al. [ 20 ], with some modifications. The LAB was cultivated, and the pellet cells were obtained by centrifugation (7000 × gfor 5 min at 4 ◦ C). After the pellets were washed twice and resuspended in PBS solution (50 mM K 2 HPO 4 /KH 2 PO 4, pH 6.5, Sigma-Aldrich, Saint Louis, MO, USA) to achieve an optical density of 0.6–0.7 (PBS containing 8 log CFU mL −1 ) (A 0 ) at 600 nm, the organic solvent n-hexadecane (Sigma-Aldrich, Saint Louis, MO, USA) was mixed (1:5) with the cell suspension and vortexed for 2 min. After 1 h of incubation at 37 ◦ C, the absorbance of the aqueous layer formed was measured at 600 nm (A). The following equation calculated cell surface hydrophobicity: Hydrophobicity (H%) = [(A0−A)/A0]×100 The values for A 0 : absorbance values measured before the extraction and A: absorbance values measured after the extraction with n-hexadecane. 2.4. Auto-Aggregation and Coaggregation Assays The isolates were grown in an MRS broth medium at 37 ◦ C for 48 h to determine specific cell–cell interactions (auto-aggregation) and with pathogenic strains (coaggregation), according to Del Re, et al. [ 21 ], with minor modifications. After incubation, the cultures were centrifuged (7000 × gfor 5 min), washed twice, and resuspended in phosphate-buffered saline (50 mM K 2 HPO 4 /KH 2 PO 4 , pH 6.5). In addition, 4 mL of the cell suspensions were mixed and incubated at room temperature for 24 h. After the incubation, absorbance at 600 nm was measured using a UV spectrophotometer (Thermo Scientific, Waltham, MA, USA) (A0). The auto-aggregation percentage was expressed as: Auto-aggregation (%) = 1 −(A0/At)×100, where A 0 represents the optical density at the beginning of the experiment and A t represents the data after 24 h. For coaggregation, LAB cultures and pathogenic microorganisms, Escherichia coli (EPEC) INCQS 00181 CDC 055 or Salmonella Enteritidis ATCC 564, were mixed in equal parts (2 mL) in pairs (LAB + selected pathogen). Control groups of individual microorganisms were tested as references. The suspensions’ absorbance at 600 nm was measured right after mixing 4 h of incubation at 37 ◦ C. Samples were retrieved following the procedure from the auto-aggregation assay. All experiments were performed in triplicate. The percentage of coaggregation was calculated using the equation by Handley, et al. [22]: Coaggregation (%) = ((Ax +Ay)/2) −A(x+y)×100, Ax +Ay/2 where xand yrepresent each of the two strains in the control tubes, and (x+y) represents the mixture. Six strains with higher values of cell surface properties were selected for the next set of assays. Fermentation 2022,8, 277 5 of 18 2.5. In Vitro Assessment of Safety Attributes 2.5.1. Gelatinase Activity Selected LAB isolates were tested for gelatinase production using a tryptone-neopeptonedextrose (TND) agar (g/L: tryptone 17.0, neopeptone 3.0, dextrose 2.5, NaCl 5.0, K 2 HPO 4 2.5 and agar 20.0, Sigma-Aldrich, Saint Louis, MO, USA) containing 0.4% gelatin. The LABs were cultivated, and 10 µ L of the cell cultures were plated in plates containing the TND medium (incubated at 37 ◦ C for 48 h). After the incubation, a saturated ammonium sulfate solution was added to flood the Petri plates. The positive reaction was defined by developing clear zones around the spots [23]. 2.5.2. DNase Production The LAB isolates were subjected to the analysis of DNase enzyme production. The strains were grown, and 10 µ L of the cell cultures were plated in a DNase agar medium (HiMedia, Einhausen, Germany). The plates were incubated at 37 ◦ C for 48 h. Then, an HCl (2 mM) solution was added enough to flood the Petri plates, and the confirmation of a DNase enzyme production was determined by a clear zone around the colonies [23]. 2.5.3. Hemolytic Activity The strains were also tested for hemolytic activity; 10 µ L of the LAB cell cultures was inoculated on a culture medium of tryptone soy agar 10% (TSA, bacto ™ Bd, Franklin Lakes, NJ, USA), supplemented with 5% (v/v) of defibrated ram’s blood. The plates were incubated for 48 h at 37 ◦ C. The positive result consisted of a clear zone of hydrolysis around the colonies. Therefore, isolates with this result cannot be selected for potential probiotic use [24]. 2.6. Antibacterial Activity Antibacterial activity was evaluated following the methodology described by Prado, et al. [ 25 ] with modifications. The LAB cells were centrifuged (7000 × gfor 5 min at 4 ◦ C), and the supernatant (pH adjusted to 7.0) was used for the diffusion technique in wells. An amount of 50 µ L was added to the wells on a BHI medium containing 1 mL of the pathogen. The plates were incubated at 37 ◦ C for 48 h, and the positive control was a culture medium without microorganisms. Growth-free inhibition zones around the well confirmed the antibacterial activity. 2.7. Adhesion of LAB Strains to Caco-2 Cell Lines 2.7.1. Growth and Maintenance of Caco-2 Cells The Caco-2 cells supplied by the Rio de Janeiro Cell Bank (BCRJ, Rio de Janeiro, Brazil) were subcultured in 24-well tissue culture plates (Sarstedt, Germany) to a 2 × 10 5 cells/mL. The medium used was a modified minimal essential (MEM) by Eagle, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 1 × non-essential amino acids, and 0.1 mg/mL gentamicin (Invitrogen, Gibco, Naerum, Denmark). Cultivation took 21 days (at 37 ◦ C, in a humidified atmosphere of 5% CO 2 ). The cell medium was changed on alternate days until cells obtained subconfluence (80–90%) and were then sub-passed [19]. 2.7.2. Adhesion to Caco-2 Cell Line The human colon adenocarcinoma cell line (Caco-2) adhesion test was performed according to the methodology used by Ramos, Thorsen, Schwan, and Jespersen [ 19 ], with certain modifications. Selected LAB isolates were cultivated (24 h, 37 ◦ C) in MRS broth and washed twice with phosphate-buffered solution (10 mM K 2 HPO 4 /KH 2 PO 4 and 150 mM NaCl, pH 7.4). Then, they were resuspended in Eagle’s MEM at an approximate concentration of 8 log CFU mL −1 . For the assay, 1 mL of the suspension of each bacterium was incubated in each well together with the cell line culture (90 min, 37 ◦ C, 5% CO 2 atmosphere). Next, washed with 1 mL of PBS three times to remove non-adherent bacteria cells, and the cell line cultures were lysed with 1 mL of Triton-X solution (0.1% v/vin Fermentation 2022,8, 277 6 of 18 PBS, 10 min, 37 ◦ C). Then, serial dilutions of the solution with released bacterial cells were performed and enumerated on MRS agar (37 ◦ C, 48 h). The percentage of initial bacteria and the counts after washings (log CFU mL −1 ) were used to calculate the adhesion capacity. The assays were repeated twice and performed in triplicate. 2.8. Survival of LAB during In Vitro Digestion The LAB that showed higher adhesion to the Caco-2 cell line (L. brevis CCMA1762, L. pentosus CCMA1768, and L. paracasei CCMA1770) were selected and exposed to simulated gastrointestinal conditions according to de Madureira, et al. [ 26 ] to evaluate their probiotic potential. The test was carried out in an incubator at 37 ◦ C with mechanical agitation ( TE-424 TECNAL, Orbital Shaker Incubator, São Paulo, Brazil) to simulate peristaltic movements with rotation adjustment in each tested phase (esophagus–stomach, duodenum, and ileum). The stages of digestion were simulated—oral phase: α -amylase solution (100 U/mL 0.1 M CaCl 2 ) pH 6.9; esophagus–stomach: pepsin (25 mg/mL diluted in 0.1 M HCl), at a rate of 0.05 mL/mL, for 90 min at 130 rpm, and in this step, the pH was gradually reduced using 1 M HCl solution (Mainville et al., 2005); duodenum: pancreatin (1 g/L diluted in 0.1 M NaHCO 3 ) and bile salts (6 g/L diluted in 0.1 M NaHCO 3 ), with a proportion of 0.25 mL/mL in the system, pH 5.0, for 30 min, at 45 rpm (Laurent et al., 2007); and ileum: 6.5 pH, using 0.1 mM/L NaHCO 3 , for 60 min, at 45 rpm. All enzymes and bovine bile salts were supplied by Sigma Aldrich (St. Louis, MO, USA). After each simulation phase, serial dilutions were performed in a sterile saline solution (0.9% NaCl) and inoculated MRS medium to enumerate viable cells (log CFU mL−1). 2.9. Antifungal Activity The strains L. brevis CCMA1762, L. pentosus CCMA1768, and L. paracasei CCMA1770 were evaluated against the fungi Aspergillus flavus MUM 08.201 and Penicillium nordicum MUM 08.16. The LAB cells were centrifuged (7000 × gfor 5 min at 4 ◦ C) followed by filtration (0.2 µ m, polypropylene), and the supernatant was stored at 4 ◦ C and used in the assays. All experiments were performed in duplicate. For antifungal analysis, the “poisoned food technique” was used according to Guimarães, et al. [ 27 ] with some modifications. The LAB supernatant was added to the MEA medium (adjusted final composition) in proportions of 25% and poured into plates. After the medium was solidified, 10 µ L of the fungal spore suspension (10 6 spores/mL) was inoculated in the center of the plates. Controls were performed without the addition of supernatant. The plates were incubated for 7 days in the dark at 25 ◦ C, and every day the diameters of the fungal colonies were measured, and the percentage of inhibition determined was determined (% Microbial inhibition = [1 − (MEA with LAB supernatant diameter/MEA control diameter)] ×100). 2.10. Aflatoxin B1 and Ochratoxin A Removal by Selected Lactic Acid Bacteria The strains L. brevis CCMA1762, L. pentosus CCMA1768, and L. paracasei CCMA1770 LAB to eliminate AFB1 and OTA mycotoxins were evaluated by Taheur, et al. [ 28 ] with some modifications. Standard stock solutions of Aflatoxin B1 and Ochratoxin A (Sigma-Aldrich, Saint Louis, MO, USA) were prepared in methanol at 2 mg/mL. For each bacterium, 5 mL of MRS broth was contaminated with mycotoxins (2 µ g/mL) and inoculated with 0.1 mL of the inoculum of already grown bacteria. Negative controls were prepared without inoculation of bacteria. After incubation (37 ◦ C, 7 days), the adsorption of mycotoxins AFB1 and OTA were tested, the tubes were centrifuged (7000 × gfor 5 min), and 2 mL of the supernatant was added and vortexed into a solution of acetonitrile/methanol/acetic acid (78/20/2, v/v/v) (Fisher Scientific, Waltham, MA, USA), then left overnight at room temperature in the dark. After this period, all samples were filtered (0.2 µ m, polypropylene) and preserved at −20 ◦C until analyzed by HPLC. Fermentation 2022,8, 277 7 of 18 2.11. Mycotoxins Analysis High-performance liquid chromatography (HPLC) with fluorescence detection was used to quantify AFB1 and OTA mycotoxins (Varian Prostar 210 pump system, Varian Prostar 410 autosampler, Jasco FP-920 fluorescence detector, and Jones Chromatography 7971 column heater—30 ◦ C). The system was operated by Varian 850-MIB data and Galaxie system chromatography data. The YMC-Pack ODS-AQ analytical C18 column (250 ×4.6 mm I.D., 5 µm) and respective pre-column were used. The mobile phase deionized water/acetonitrile/methanol (3/1/1, v/v/v, 1.0 mL/min) was used for the detection of AFB1, after post-photochemical column derivatization (PHRED unit-Aura Industries, San Diego, CA, USA, ex: 365 nm, in: 435 nm) [ 29 ]. The mobile phase acetonitrile/water/acetic acid (99/99/2, v/v/v, 1.0 mL/min) was used for the detection of OTA, with ex: 333 nm and at: 460 nm Abrunhosa, et al. [ 30 ]. For the detection, the injection volume was 50 µ L for both mycotoxins. Standard curves were prepared for the quantification of mycotoxins, with concentrations ranging from 0.005 to 1.0 µ g/mL. AFB1: LOD = 0.004 µ g/mL and LOQ = 0.013 µ g/mL, mean recovery 93 ± 7.0%, OTA: LOD = 0.005 µg/mL and LOQ = 0.017 µg/mL, mean recovery f 113 ±6.7%. 2.12. Statistical Analyses The SISVAR 5.1 software [ 31 ] was used to analyze variance and the Scott–Knott test. Differences in results were considered statistically significant when p< 0.05. 3. Results 3.1. Screening of LAB Strains The pre-selection of LAB cultures was first based on their ability to survive at low pH levels and in the presence of bile salts. Twelve LAB strains showing the highest survival rate in this assay were selected and evaluated for cell surface characteristics (hydrophobicity, self-aggregation, and coaggregation with pathogens) and safety attributes. Six strains with higher values concerning cell surface properties and safety were selected as probiotic candidates for further characterization by antimicrobial activity and adhesion capacity to Caco-2 cells. Finally, three bacteria were selected for survival during in vitro digestion. 3.2. Acid and Bile Salt Tolerance Fourteen strains obtained from naturally fermented table olive were exposed to acidic conditions and the presence of bile salts at 37 ◦ C (Table 2). After 3 h of incubation, twelve strains showed tolerance under these conditions, maintaining survival at pH 2 and at a concentration of 0.3% bile salts (>6 log CFU mL −1 ), which is comparable or statistically similar (p> 0.05) to the reference strain L. paracasei LBC-81 (>7.79 log CFU mL −1 after 3 h of incubation) (Table 2). The strains L. paracasei CCMA1775 and L. paracasei CCMA1764 showed reduced viability (p< 0.05) at pH 2 and a 0.3% concentration of bile salts after 3 h of incubation (<4.5 log CFU mL −1 ) and were not considered in the following tests. The other isolates had a high survival rate ranging from 84.38 to 100% in both exposure conditions. Fermentation 2022,8, 277 8 of 18 Table 2. Viability of LAB isolates after exposure to acid and bile salt conditions. Acid Condition Time of Exposure (h) Bile Salt Condition Time of Exposure (h) LAB Strains T0 T3 Survival (%) T0 T3 Survival (%) L. brevis CCMA1766 7.92 ±0.01 aB 9.15 ±0.34 aA 100 7.67 ±0.16 aA 8.35 ±0.07 aA 100 L. paracasei CCMA1763 7.52 ±0.02 aA 7.97 ±0.04 bA 100 7.42 ±0.02 aB 8.81 ±0.16 aA 100 L. paracasei CCMA1764 7.86 ±0.02 aA 4.25 ±0.49 dB 54.07 7.80 ±0.06 aA 4.09 ±0.16 cB 52.43 L. pentosus CCMA1768 7.65 ±0.04 aA 7.88 ±0.14 bA 100 7.42 ±0.06 aA 7.38 ±0.45 bA 99.44 L. paracasei CCMA1769 7.63 ±0.04 aA 7.73 ±0.04 bA 100 7.73 ±0.04 aA 7.70 ±0.05 aA 99.65 L. paracasei CCMA1770 7.88 ±0.04 aA 7.41 ±0.34 bA 94.13 7.59 ±0.08 aA 8.23 ±0.11 aA 100 L. brevis CCMA1762 7.96 ±0.03 aA 6.73 ±0.05 cB 84.55 7.56 ±0.09 aA 8.36 ±0.48 aA 100 L. paracasei CCMA1772 7.65 ±0.06 aA 6.72 ±0.03 cB 87.88 7.75 ±0.05 aA 7.74 ±0.19 aA 99.86 L. paracasei CCMA1771 7.85 ±0.13 aA 6.64 ±0.06 cB 84.56 7.84 ±0.01 aA 8.44 ±0.41 aA 100 L. brevis CCMA1765 7.53 ±0.08 aA 6.73 ±0.05 cB 89.39 7.86 ±0.02 aA 7.85 ±0.16 aA 99.87 L. paracasei CCMA1767 7.89 ±0.02 aA 6.76 ±0.08 cB 85.64 7.79 ±0.12 aA 7.55 ±0.27 aA 97.02 L. paracasei CCMA1773 7.87 ±0.04 aA 6.64 ±0.06 cB 84.38 7.81 ±0.03 aA 7.91 ±0.10 aA 100 L. paracasei CCMA1774 7.77 ±0.05 aA 6.80 ±0.16 cB 87.49 7.80 ±0.06 aB 8.64 ±0.29 aA 100 L. paracasei CCMA1775 7.65 ±0.06 aA 4.04 ±0.06 dB 52.81 7.62 ±0.06 aA 4.02 ±0.05 cB 52.76 L. paracasei LBC-81 7.91 ±0.02 aA 7.79 ±0.12 bA 98.48 7.63 ±0.06 aA 8.60 ±0.18 aA 100 Results Log CFU mL −1 are expressed as mean ± SD, determined in duplicate. Mean values of different letters, lower case in columns and uppercase in rows differ significantly (p< 0.05) by the Scott–Knott test. Time of exposure: T0 : Initial mean count; T3 : Mean count after 3 h. Survival at pH 2 (%) or bile salt (%) = Final viable count (Log CFU mL −1 )/Initial viable count (log CFU mL −1 )) × 100. Strains showing potential properties as probiotics used during the simulated digestion are bolded. 3.3. Cell Surface Hydrophobicity Microbial adhesion to non-polar solvents (n-hexadecane) reflects cell surface hydrophobicity. In this study, L. brevis CCMA1762 (31.23 ± 1.7%), L. paracasei CCMA1768 ( 28.13 ±2.1% ), and L. paracasei CCMA1770 (27 ± 0.7%) showed higher (p< 0.05) adhesion to the solvent n-hexadecane and are, therefore, able to interact with more cell bodies compared to other strains, similar (p> 0.05) to the hydrophobicity of reference strain L. paracasei LBC-81 (29.59 ± 0.9%). For the other isolates, the percentage of hydrophobicity ranged from 15.4 ±1.4 to 24.03 ±1.2 % (Figure 1). Fermentation2022,8,xFORPEERREVIEW9of18    Figure1.Percentage(%)ofHydrophobicityobtainedforthedifferentLABisolates.Barindicates SD.Barsfollowedbydifferentlettersdiffersignificantly(p<0.05)bytheScott – Knotttest. 3.4.Auto‐AggregationandCoaggregationofthePathogens’Ability Auto‐aggregationiscloselyassociatedwithadhesion[32].Bothareresponsiblefor colonizationintheGIT;however,coaggregationisassociatedwithinteractionwithpath‐ ogenicbacteria.Allstrainsshowedahighpercentageofauto‐aggregation(>60%)after24 hofincubation(Figure2).Amongthepotentialprobioticstrains,L.pentosusCCMA1768 (88.83±1.7%),L.paracaseiCCMA1771(84.75±0.7%),L.paracaseiCCMA1766(84.77±2.1%), L.paracaseiCCMA1770(81.85±1.6%),L.brevisCCMA1762(81.38±2.1%),andL.paracasei CCMA1774(80.34±0.7%)providedauto‐aggregationpercentagessimilar(p>0.05)tothat ofthereferencestrain(81.86±2.1%).Theotherisolates,despiteshowingalowerpercent‐ ageofauto‐aggregation(p<0.05)comparedtothereferencestrain,stillshowedrelatively highvaluesofauto‐aggregation,rangingfrom74.07±1.6to79.08±2.3%.  Figure2.Percentageofauto‐aggregationofLABisolatesBarindicatesSD.Barsfollowedbydifferent lettersdiffersignificantly(p<0.05)bytheScott – Knotttest. Figure 1. Percentage (%) of Hydrophobicity obtained for the different LAB isolates. Bar indicates SD. Bars followed by different letters differ significantly (p< 0.05) by the Scott–Knott test. 3.4. Auto-Aggregation and Coaggregation of the Pathogens’ Ability Auto-aggregation is closely associated with adhesion [ 32 ]. Both are responsible for colonization in the GIT; however, coaggregation is associated with interaction with pathogenic bacteria. All strains showed a high percentage of auto-aggregation (>60%) after 24 h of incubation (Figure 2). Among the potential probiotic strains, L. pentosus Fermentation 2022,8, 277 9 of 18 CCMA1768 ( 88.83 ±1.7% ), L. paracasei CCMA1771 (84.75 ± 0.7%), L. paracasei CCMA1766 ( 84.77 ±2.1% ), L. paracasei CCMA1770 (81.85 ± 1.6%), L. brevis CCMA1762 (81.38 ± 2.1%), and L. paracasei CCMA1774 (80.34 ± 0.7%) provided auto-aggregation percentages similar (p> 0.05) to that of the reference strain (81.86 ± 2.1%). The other isolates, despite showing a lower percentage of auto-aggregation (p< 0.05) compared to the reference strain, still showed relatively high values of auto-aggregation, ranging from 74.07 ± 1.6 to 79.08 ±2.3%. Fermentation2022,8,xFORPEERREVIEW9of18    Figure1.Percentage(%)ofHydrophobicityobtainedforthedifferentLABisolates.Barindicates SD.Barsfollowedbydifferentlettersdiffersignificantly(p<0.05)bytheScott – Knotttest. 3.4.Auto‐AggregationandCoaggregationofthePathogens’Ability Auto‐aggregationiscloselyassociatedwithadhesion[32].Bothareresponsiblefor colonizationintheGIT;however,coaggregationisassociatedwithinteractionwithpath‐ ogenicbacteria.Allstrainsshowedahighpercentageofauto‐aggregation(>60%)after24 hofincubation(Figure2).Amongthepotentialprobioticstrains,L.pentosusCCMA1768 (88.83±1.7%),L.paracaseiCCMA1771(84.75±0.7%),L.paracaseiCCMA1766(84.77±2.1%), L.paracaseiCCMA1770(81.85±1.6%),L.brevisCCMA1762(81.38±2.1%),andL.paracasei CCMA1774(80.34±0.7%)providedauto‐aggregationpercentagessimilar(p>0.05)tothat ofthereferencestrain(81.86±2.1%).Theotherisolates,despiteshowingalowerpercent‐ ageofauto‐aggregation(p<0.05)comparedtothereferencestrain,stillshowedrelatively highvaluesofauto‐aggregation,rangingfrom74.07±1.6to79.08±2.3%.  Figure2.Percentageofauto‐aggregationofLABisolatesBarindicatesSD.Barsfollowedbydifferent lettersdiffersignificantly(p<0.05)bytheScott – Knotttest. Figure 2. Percentage of auto-aggregation of LAB isolates Bar indicates SD. Bars followed by different letters differ significantly (p< 0.05) by the Scott–Knott test. The coaggregation percentage of the LAB against S. Enteriditis and E. coli was evaluated for the selected bacteria (Figure 3a,b). The following strains showed a higher ( p< 0.05 ) coaggregation percentage with the pathogen S. Enteriditis: L. paracasei CCMA1771 ( 63.6 ±3.4% ), L. pentosus CCMA1768 (64.79 ± 2%), and L. brevis CCMA1762 (63.02 ± 1%). The coaggregation rates of the remaining strains with the pathogen S. Enteriditis ranged from 28.43 ± 0.5 to 56.09 ±0.6%, whereas the reference strain L. paracasei LBC-81 was 54 ±0.9%. Fermentation2022,8,xFORPEERREVIEW10of18   ThecoaggregationpercentageoftheLABagainstS.EnteriditisandE.coliwaseval‐ uatedfortheselectedbacteria(Figure3,b).Thefollowingstrainsshowedahigher(p< 0.05)coaggregationpercentagewiththepathogenS.Enteriditis:L.paracaseiCCMA1771 (63.6±3.4%),L.pentosusCCMA1768(64.79±2%),andL.brevisCCMA1762(63.02±1%). ThecoaggregationratesoftheremainingstrainswiththepathogenS.Enteriditisranged from28.43±0.5to56.09±0.6%,whereasthereferencestrainL.paracaseiLBC‐81was54± 0.9%.  Figure3.Percentage(%)coaggregationofLABisolateswithpathogenicmicroorganisms:(a):coag‐ gregationwithSalmonellaEnteritidisATCC564;(b):coaggregationwithEscherichiacoli(EPEC)CDC 055.BarindicatesSD.Barsfollowedbydifferentsuperscriptlettersdiffersignificantly(p<0.05)by theScott – Knotttest. Allselectedstrainswereabletoco‐aggregatewiththepathogenE.coli,presenting coaggregationrateshigher(p<0.05)thanthereferencestrainL.paracaseiLBC‐81(22.37± 1.7%).Valuesrangedfrom25.6±0.5%forL.brevisCCMA1765to42.95±1.2%forL.para‐ caseiCCMA1770. 3.5.InVitroAssessmentofSafetyAttributes Theisolatestestedinthisstudydidnotappearpositiveforgelatinase,DNase,and hemolyticactivity(datanotshown),validatingtheirrelativesafetyasprobioticcandi‐ dates. 3.6.AntibacterialActivity Theinhibitoryactivityofstrainsagainstthepathogenicbacteriawasrecordedforsix bacteriathatshowedthebestresultsforcellsurfacetestsandweresafetobeusedaspro‐ biotics.Theinhibitionhaloswereintherangeof4.3(+)–9mm(+++)(Table3).Allthe strainsshowedantagonisticactivityagainstalltestedpathogens,withS.aureusATCC 8702beingthemostsensitivetotheeffectsofLAB,inwhichaclearlydefinedzoneof inhibition≥8mmwasobservedinthecell‐freesupernatantwellsofL.paracasei CCMA1774,CCMA1770,andL.brevisCCMA1766bacteria.Suchhighinhibitionrates werealsoobservedforotherpathogenicmicroorganisms—thesupernatantoftheL.para‐ caseiCCMA71bacteriumshowedgreaterinhibitionhalosforthepathogenS.Enteritidis ATCC564,andthesupernatantofL.brevisCCMA1762andL.pentosusCCMA1768bacte‐ riashowedgreaterefficiencyagainstthepathogenL.monocytogenesATCC19117. Figure 3. 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