Heliyon 9 (2023) e21558 Available online 3 November 2023 2405-8440/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Assessment of probiotic properties of lactic acid bacteria isolated from an artisanal Colombian cheese Samantha Roldán-Pérez a, Sara Lucía Gómez Rodríguez a, José Uriel Sepúlveda- Valencia a, Orlando Simón Ruiz Villadiego a, María Elena Márquez Fernández a, Olga I. Montoya Campuzano a, Mónica María Durango-Zuleta b,* a Universidad Nacional de Colombia sede Medellín, Faculty of Agricultural Sciences, Medellín, Colombia b Institución Universitaria Colegio Mayor de Antioquia, Faculty of Agricultural Sciences, Medellín, Colombia A R T I C L E I N F O Keywords: Lactic acid bacteria Autochthonous food Probiotics Double-cream cheese Foodborne pathogens Exopolysaccharide A B S T R A C T Lactic Acid Bacteria play an important role in the milk fermentation processes of traditional cheeses and have become an important target for the development of novel cheese cultures because of their ability to confer health benefits. This study aimed to evaluate the probiotic potential of 12 Lactic Acid Bacteria (LAB) strains previously isolated and molecularly identified from an artisanal Colombian Double-Cream Cheese. Probiotic properties, including safety (he molysis and sensibility to antibiotics), pH and bile salt tolerance, auto-aggregation, cell surface hydrophobicity, antibacterial activity, and exopolysaccharide production, were examined. None of the strains were hemolytic, and Pediococcus (16, 18) and Lactobacillus (28, 29) were found to be sensitive to all antibiotics. Moreover, all the strains tolerated pH (3.0, 6.5 and 8.0) and bile salt conditions (0.3, 0.6 and 1.0 % w/v). Pediococcus pentosaceus (16), Leuconostoc citreum (17), Pediococcus acidilactici (18), Enterococcus faecium (21,22), Enterococcus faecalis (24) and Limosi lactobacillus fermentum (29) exhibited medium autoaggregation and affinity to chloroform. Six of the strains exhibited a ropy exopolysaccharide phenotype. Antibacterial activity against food borne pathogens, Salmonella Typhimurium ATCC 14028, Listeria monocytogenes ATCC 19111, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, was found to be strain dependent, with the strains 16, 18, 21, 26, 28 and 29 presenting a higher inhibition (>4 mm) against all of them. According to Principal Component Analysis, P. pentosaceus (16), Leu. mes enteroides (26), L. casei (28), L. fermentum (29), and E. faecium (21) showed strong probiotic properties. Our findings suggest that five strains out of the 12 sampled strains are potential probiotics that could be used in the processing of traditional dairy products on an industrial scale to improve their quality. 1. Introduction Fermented foods with probiotics have been proposed as a novel approach for the therapeutic treatment of comorbidities of COVID- * Corresponding author. Institución Universitaria Colegio Mayor de Antioquia, Faculty of Health Sciences. Medellín, Colombia. E-mail addresses: sroldanp@unal.edu.co (S. Roldán-Pérez), slgomezr@unal.edu.co (S.L. Gómez Rodríguez), jusepul@unal.edu.co (J.U. Sepúlveda-Valencia), osruiz@unal.edu.co (O.S. Ruiz Villadiego), memarque@unal.edu.co (M.E. Márquez Fernández), oimontoy@unal.edu. co (O.I. Montoya Campuzano), monica.durango@colmayor.edu.co (M.M. Durango-Zuleta). Contents lists available at ScienceDirect Heliyon journal homepage: www.cell.com/heliyon https://doi.org/10.1016/j.heliyon.2023.e21558 Received 10 May 2023; Received in revised form 18 October 2023; Accepted 24 October 2023 mailto:sroldanp@unal.edu.co mailto:slgomezr@unal.edu.co mailto:jusepul@unal.edu.co mailto:osruiz@unal.edu.co mailto:memarque@unal.edu.co mailto:oimontoy@unal.edu.co mailto:oimontoy@unal.edu.co mailto:monica.durango@colmayor.edu.co www.sciencedirect.com/science/journal/24058440 https://www.cell.com/heliyon https://doi.org/10.1016/j.heliyon.2023.e21558 https://doi.org/10.1016/j.heliyon.2023.e21558 http://crossmark.crossref.org/dialog/?doi=10.1016/j.heliyon.2023.e21558&domain=pdf https://doi.org/10.1016/j.heliyon.2023.e21558 http://creativecommons.org/licenses/by-nc-nd/4.0/ Heliyon 9 (2023) e21558 2 19 disease (SARS-CoV-2) due to their ability to modulate the immune system and intestinal microbiome and reduce susceptibility to subsequent infections [1–3]. Currently, probiotics have been used to treat or prevent some pathologies such as Crohn’s disease, ul cerative colitis, acute respiratory tract infections, necrotizing enterocolitis, food allergies, slow the progression of liver fibrosis, irri table bowel syndrome, type II diabetes, and obesity [4–8]. The benefits of the consumption of probiotics have been found to be directly related to their effect on the gut microbiota, which varies according to genetics, age, ethnicity, and the geographic origin of the host and diet [9]. Also, they may contribute to consumer health by impacting mucosal immune mechanisms and gut microbiota and their influence on other organs of the body through the production of neurotransmitters [10,11]. In traditional cheeses, Lactic Acid Bacteria (LAB) with probiotic properties play an important role when added accidently or intentionally [12]. During milk fermentation, the genera Lactobacillus, Lactococcus, Streptococcus, and Enterococcus have shown the ability to reduce the concentration of lactose, facilitate the elimination of toxic or antinutritional factors from milk, promote better bioavailability of nutrients, and prevent adverse reactions from their consumption [13]. For LAB to be considered a probiotic, they must meet the requirements established by FAO/WHO which include being safe for consumption, not being hemolytic, resisting the acidic pH of the stomach, bile salts, and gastric juices, being sensitive to antibiotics, having antagonistic activity against pathogens, having autoaggregation, hydrophobicity and adhesion to intestinal epithelium [14]. In addition, new selection criteria include bio logical activities such as anti-carcinogenic, anti-depressant, anti-obesity, antidiabetic, and hypocholesterolemic activities [15]. In Colombia, various studies have been carried out on probiotics for functional food and animal feed design, including techniques for supplementing plant-based matrices with microorganisms [16–18]. However, commercial strains are often used because of the presence of unknown microbiota in traditional foods [19]. Fresh cheese is the most consumed artisan cheese in Colombia [20] and has a microbiological composition with beneficial properties [21]. Therefore, it is important to study the microbiota of indigenous Colombian foods, such as artisan cheeses, as they could provide new native strains with probiotic properties for developing innovative dairy products with positive effects on human health [22]. Cheese is considered a better carrier of probiotics compared to many dairy products, due to its lower water activity (aw > 0.90), low storage temperature (4–8 ◦C), relatively higher pH compared to traditional dairy products, and textural characteristics (e.g., high fat content, which protects probiotics during gastrointestinal transit) [23]. The aim of this work was to evaluate the probiotic properties of twelve LAB strains previously isolated from a Colombian Double Cream Cheese and select the most promising probiotic strains that could give additional value to the manufacture of traditional dairy products and new, innovative, and precision foods. 2. Material and methods 2.1. Strains and culture conditions Twelve LAB strains previously isolated and molecularly identified from a Colombian Double Cream Cheese [21] were evaluated for their probiotic properties: Pediococcus pentosaceus (16), Pediococcus acidilactici (18), Leuconostoc citreum (17), Leuconostoc mesenteroides subsp. mesenteroides (19, 26), Weissella viridescens (25), Lacticaseibacillus casei (28), Limosilactobacillus fermentum (29), Lactococcus lactis (27), Enterococcus faecium (21, 22) and Enterococcus faecalis (24). The strains were used under the permission number 16DE0AC88E5 given by the Autoridad Nacional de Licencias Ambientales (ANLA). As a positive control, L. rhamnosus GG ATCC 53103 was used. Bacteria were cultivated in de Man, Rogosa, and Sharpe (MRS) broth (Merck, Darmstadt, Germany) at 37 ◦C in an atmosphere with 5 % CO2 and 95 % air. All the assays were carried out starting from a bacterial solution adjusted to 1.5 × 108 Colony Forming Units (CFU/mL). Briefly, cells were harvested by centrifugation at 3500g for 5 min. After washing the pellet, the cell suspension was adjusted in Phosphate Buffered Saline to reach a concentration 0.5 McFarland. 2.2. Biosafety assessment 2.2.1. Hemolytic activity Hemolytic activity was evaluated using blood agar with 5 % (v/v) human blood. Bacterial overnight cultures in MRS broth were streaked on the surface of the agar, and the plates were incubated at 37 ◦C for 48 h [24]. Bacteria were classified according to the halo produced around the inoculation line. Lacticaseibacillus rhamnosus GG ATCC 53103 and Staphylococcus aureus coagulase positive ATCC 25923 were used as negative and positive controls, respectively. 2.2.2. Antibiotic susceptibility assessment Minimal Inhibitory Concentrations (MICs) of all bacteria to gentamicin, penicillin, chloramphenicol, ampicillin, erythromycin, and vancomycin (Sigma) were determined by the microdilution broth method according to the CLSI (Clinical and Laboratory Standards Institute) and EFSA (European Food Safety Authority) [25,26]. 100 μL of the bacterial inoculum (0.5 McFarland) was added to each well of a 96-well plate containing 100 μL of an increasing concentration of the antibiotics. The microplates were incubated at 37 ◦C for 24 h, and bacterial MICs were determined as the lowest concentration of an antibiotic at which visible growth was inhibited and compared with the MIC breakpoint values recommended by EFSA [26]. 2.3. Screening of probiotic properties 2.3.1. Tolerance to pH and bile salts Tolerance to pH (3.0, 6.5, and 8.0) and bile salts (0.3, 0.6, and 1.0 % (w/v)) was assessed using the broth microdilution method in S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 3 96-well plates, and modified MRS broth solutions were prepared as indicated by Jatmiko [27]. Stock solutions of MRS broth were prepared and adjusted to each of the pH values using HCl and NaOH at 1 mol/L, and the appropriate amount of bovine bile salts was added according to the concentration (Difco Oxgall). Then, 100 μL of each strain (0.5 McFarland) was mixed with the previously prepared MRS broth solutions. The plates were incubated at 37 ◦C for 24 h in the Multiskan™ GO Thermo spectrophotometer (Thermo Scientific), and the Optical Density (OD) at 600 nm was recorded every hour. To confirm the tolerance to the extreme conditions (pH 3.0 and 1 % (w/v) bile salts), the strains were adjusted to the 0.5 McFarland standard in peptone water (1 % (w/v)), mixed with the previously prepared MRS broth solutions and incubated at 37 ◦C for 2 h in anaerobic conditions. Serial dilutions were performed, and CFU/mL counts were made by the standard plate count method on MRS agar. Tolerance to these conditions was expressed as Log (CFU/mL) for each strain [28]. 2.3.2. Auto-aggregation and cell surface hydrophobicity The autoaggregation of the LAB strains was assessed according to the method of Armas [29], and the cell surface hydrophobicity was determined using the method described by Lee [30] with small modifications. In both cases, the cells from an overnight culture of each strain were harvested by centrifugation. After washing the pellet, it was resuspended in phosphate-buffered saline (pH 7.2) and the absorbance was adjusted to 0.1 OD600nm (A0, H0). For the auto-aggregation assay, each bacterial suspension was incubated at 37 ◦C without agitation, and the absorbance of the surface was measured at 2h (A1) at 600 nm using a UV/visible spectrophotometer. Autoaggregation was expressed as %Autoaggregation = (1-(A1)/(A0)) × 100. For hydrophobicity, the bacterial suspensions were mixed with chloroform (a non-polar acid solvent) or ethyl acetate (a non-polar basic solvent) at a 2:1 ratio and vortexed for 1 min. The mixture was incubated at ambient temperature for 1 h until phase separation. Briefly, the aqueous phase was slightly removed to measure its absorbance at 600 nm (H1). The cell surface hydrophobicity was calculated as %H = (1-(H1)/(H0)) × 100. 2.3.3. Exopolysaccharide (EPS) production phenotype EPS production was evaluated according to the procedure described by Khalil [31]. Briefly, strains were inoculated on the surface of MRS agar supplemented at 2 % (w/v) with sucrose (Merck), and bacterial plates were incubated at 37 ◦C for 48 h. Subsequently, a smear was made in one of the colonies with an inoculation loop, and the formation of an unbreakable strand indicated a ropy phenotype. 2.3.4. Antibacterial activity against foodborne pathogens To evaluate the antibacterial activity of LAB against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, Listeria monocytogenes ATCC 19111, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923, the agar well diffusion method was performed using Mueller Hinton Agar (MHA) or Trypticase Soy Agar (TSA) (Merck) plates [32]. To obtain cell-free supernatant, overnight cultures of LAB until stationary phase were centrifuged at 10000 g for 20 min at 4 ◦C and sterilized with a filter membrane of 0.22 μm. Briefly, the pathogenic strains were incubated in Brain Heart Infusion (BHI) medium (Merck) at 37 ◦C for 24 h under aerobic conditions. Thereafter, the cells were harvested by centrifugation, the pellet was washed, resuspended in phosphate-buffered saline (pH 7.2), and the absorbance was adjusted to 0.1 OD600nm. 100 μL of the adjusted suspension of each pathogenic strain was grown over soft MHA plates, except for L. monocytogenes, which was inoculated in TSA. Then, wells (5 mm) were made in the agar plates and filled with 50 μL of cell free supernatant of LAB strains. Plates were incubated at 37 ◦C for 12–24 h. The antibacterial activity scores were assigned according to the diameter of the inhibition zone: Negative (− ) for inhibition zones ≤1.0 mm; Intermediate (+) for inhibition zones of 1.1–3.9 mm and High (++) for inhibition zones ≥4.0 mm [33]. 2.4. Statistical analysis The tests were performed in triplicate for each treatment. In all cases, results were expressed as mean ± standard deviation (SD), and significant differences were considered when p < 0.05 with a 95 % confidence interval. When the data were non-parametric, the Table 1 Minimum inhibitory concentration (MIC) (μg/mL). Strain Gm Pe Vm Cm Te Am Er 16 <4 <1 N.R. 4 4 <1 <0.25 17 16 <1 N.R. <4 1.5 <0.5 2 R 18 <4 <1 N.R. <2 4 <1 <0.25 19 32R <1 N.R. 16R 0.19 <0.5 2R 21 >64R <1 >64R 16 1.5 <0.5 <1 22 >64R <1 <2 16 0.125 <0.5 2 24 >64R <1 <2 16 0.125 <0.5 8R 25 16 <1 N.R. 8R 0.25 <0.5 2R 26 16 <1 N.R. 4 2 1 2R 27 32 <1 <2 8 0.094 <0.5 2R 28 8 <1 N.R. <2 4 <1 <0.25 29 8 <1 N.R. <2 4 <0.5 0.5 Gm: gentamycin, Pe: penicillin, Vm: vancomycin, Cm: chloramphenicol, Te: tetracycline, Am: ampicillin, Er: erythromycin; R: Resistant NR: not required. S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 4 Kruskal-Wallis test was performed. For the analysis of the data, a programming routine was carried out using the statistical software SPSS version 25 (IBM® SPSS® Statistics). In addition, a Principal Components Analysis (PCA) using the SPSS Statistics v.24 (IBM) program was performed to determine the most promising probiotic strains. 3. Results 3.1. Biosafety assessment All of the LAB strains were non-hemolytic. The MIC values for antibiotic susceptibility of the strains to the antibiotics are presented in Table 1. All the strains exhibited MIC values equal to or lower than the MIC breakpoints reported by EFSA [34] for penicillin, tetracycline, and ampicillin, indicating that they were sensitive to these antibiotics. P. pentosaceus (16), P. acidilactici (18), L. casei (28), and L. fermentum (29) were sensitive to all of the antibiotics tested (Ali et al., 2021). The strains L. citreum (17), L. mesenteroides subsp. mesenteroides (26), and L. lactis (27) were only resistant to erythromycin. L. mesenteroides subsp. mesenteroides (19) and Enterococcus (21, 22 and 24) presented resistance to several antibiotics. 3.2. 2. Tolerance to pH and bile salts The results of LAB tolerance at different pH values are presented in Table 2. Vigorous growth was observed in all strains at pH 6.5 and 8.0. L. casei (28), L. fermentum (29), E. faecium (21) and E. faecalis (24) showed the highest growth of all evaluated pH. At pH 3.0, no significant difference was observed (p > 0.05) in strain tolerance, compared with L. rhamnosus GG when the microdilution broth method was used. The strains 16, 18, 21, 22, 26, 28, and 29 were tolerant to 0.3 % (w/v) and 0.6 % (w/v) of bile salt, showing no significant differences (p > 0.005) compared to the control L. rhamnosus GG. The growth of all strains was lower at 0.6 % (w/v) biliary salts (Table 3). The growth of strains to pH 3.0 and 1.0 % (w/v) bile salts is presented in Fig. 1. The growth of the strains was higher than 6 Log (CFU/mL) in both conditions, without statistically significant differences when compared to the growth on MRS broth. Additionally, the strains 18, 25, and 26 presented higher growth at pH 3.0 and the strains 21, 25 and 26 showed a higher growth at 1.0 % (w/v) bile salts. These results confirm the data obtained by the microdilution broth method, affirming the strain’s tolerance to gastrointestinal stress conditions. 3.3. Autoaggregation and cell surface hydrophobicity Table 3 presents the autoaggregation and cell surface hydrophobicity percentages for all the strains. For the aggregation property, the classification proposed by Darmastuti [35] was used, according to which the strains P. pentosaceus (16), L. citreum (17), Table 2 Tolerance of strains to pH and bile salts (OD600 nm 23h). Strains pH Bile salts (% w/v) MRS 3.0 6.5 8.0 MRS 0.3 0.6 1.0 16 0.605 ± 0.00 abcdef 0.008 ± 0.00a 0.525 ± 0.03 afg 0.390 ± 0.15a 0.507 ± 0.031 abcde 0.342 ± 0.051abcd 0.236 ± 0.016 abcde 0.213 ± 0.016 abcd 17 0.409 ± 0.13 cdef 0.002 ± 0.00a 0.580 ± 0.01 abcdefg 0.544 ± 0.19a 0.550 ± 0.035 bcde 0.180 ± 0.043 de 0.149 ± 0.052 cde 0.144 ± 0.057 bcd 18 0.635 ± 0.01 abcde 0.004 ± 0.00a 0.587 ± 0.01 abcdef 0.397 ± 0.15a 1.137 ± 0.063 abc 0.360 ± 0.007 abc 0.331 ± 0.039 abc 0.255 ± 0.035 ab 19 0.206 ± 0.03f 0.003 ± 0.00a 0.557 ± 0.02 bcdefg 0.527 ± 0.27a 0.688 ± 0.185e 0.154 ± 0.018e 0.092 ± 0.009e 0.076 ± 0.017d 21 1.032 ± 0.00 ab 0.054 ± 0,00a 1064 ± 0,01 abcde 0,866 ± 0,30a 0.678 ± 0.466 abcde 0.337 ± 0.046 abcd 0.238 ± 0.105 abcde 0.180 ± 0.041 abcd 22 0,714 ± 0,18 abcde 0,003 ± 0,00a 0,644 ± 0,25 cdfg 0,599 ± 0,29a 0.762 ± 0.410 abcde 0.307 ± 0.069 abcde 0.242 ± 0.118 abcde 0.189 ± 0.063 abcd 24 0.687 ± 0.32 abcdef 0.024 ± 0.04a 0.742 ± 0.31 bcdefg 0.588 ± 0.37a 0.438 ± 0.028 de 0.203 ± 0.040 cde 0.146 ± 0.058 cde 0.125 ± 0.051 cd 25 0.387 ± 0.03 def 0.004 ± 0.00a 0.537 ± 0.04 dfg 0.498 ± 0.16a 0.516 ± 0.078 cde 0.154 ± 0.038e 0.136 ± 0.054 de 0.135 ± 0.050 bcd 26 0.484 ± 0.03 bcdef 0.000 ± 0.00a 0.601 ± 0.19 dfg 0.335 ± 0.09a 0.546 ± 0.006 abcd 0.383 ± 0.039 ab 0.256 ± 0.046 abcd 0.223 ± 0.064 abc 27 0.324 ± 0.10 ef 0.003 ± 0.00a 0.551 ± 0.01 defg 0.240 ± 0.14a 0.576 ± 0.091 abcde 0.241 ± 0.096 bcde 0.196 ± 0.065 bcde 0.177 ± 0.055 abcd 28 1.024 ± 0.05a 0.024 ± 0.04a 1.069 ± 0.01 abe 0.811 ± 0.26a 1.122 ± 0.005a 0.324 ± 0.047 abcde 0.363 ± 0.039 ab 0.264 ± 0.006a 29 0.963 ± 0.14 abc 0.010 ± 0.01a 1.074 ± 0.01 ab 0.863 ± 0.30a 1.131 ± 0.015a 0.326 ± 0.053 abcd 0.390 ± 0.030a 0.275 ± 0.009a Control Rha 1.072 ± 0.04a 0.161 ± 0.28a 1.209 ± 0.03a 0.858 ± 0.44a 1.169 ± 0.050a 0.657 ± 0.035a 0.435 ± 0.036a 0.292 ± 0.049a Means in the same column without a common superscript letter differ significantly (P < 0.05). Rha, L. rhamnosus GG. S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 5 Table 3 Autoaggregation and hydrophobicity percentages. Strain % Autoaggregation % Hydrophobicity Ethyl acetate Chloroform 16 21.49 ± 4.46 abcd 31.89 ± 3.79 abcde 57.81 ± 3.80 abc 17 23.77 ± 3.49 ab 46.48 ± 0.86 a 53.98 ± 1.52 abcd 18 35.76 ± 3.37 a 24.44 ± 2.37 bcde 18.59 ± 2.34 cd 19 19.27 ± 2.45 bcde 18.90 ± 2.91 cde 66.85 ± 3.62 ab 21 20.32 ± 4.59 bcde 15.99 ± 1.23 de 44.93 ± 5.15 abcd 22 20.14 ± 1.80 abcde 30.50 ± 1.44 abcde 17.66 ± 4.23 cd 24 21.27 ± 2.42 abc 18.91 ± 0.12 cde 13.74 ± 3.87 d 25 14.09 ± 3.57 e 41.97 ± 2.19 ab 30.71 ± 0.26 abcd 26 23.30 ± 7.25 abcd 35.99 ± 4.57 abc 69.74 ± 11.28 ab 27 15.32 ± 3.37 cde 36.25 ± 0.38 abc 22.30 ± 1.35 bcd 28 15.77 ± 3.82 cde 28.28 ± 3.00 abcde 42.57 ± 4.75 abcd 29 21.82 ± 2.41 abc 14.03 ± 3.98 e 87.55 ± 0.43 a ATCC 53103 15.91 ± 1.78 de 33.31 ± 0.44 abcd 87.20 ± 2.55 a Fig. 1. Viability of LAB strains to pH and bile salts conditions. Fig. 2. EPS producing LAB. A. P. pentosaceus (16), B. P. acidilactici (18), C. E. faecium (21), D. W. viridescens (25), E. L. casei (28) and F. L. fermentum (29). S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 6 P. acidilactici (18), E. faecium (22), E. faecalis (24), L. mesenteroides (26), and L. fermentum (29) presented medium autoaggregation (20–70 %), and the other strains were classified with low autoaggregation (<20 %). Strains were classified as having low (0–30 %), medium (31–60 %) or high (>60 %) hydrophobicity. For ethyl acetate, the strains 16, 17, 25, 26 and 27 were classified in the medium hydrophobicity range without statistical differences (p > 0.05) with the strains 24 and 28; the remaining strains were classified as having low hydrophobicity. For chloroform, 41.6 % of strains were classified as low hydrophobic, 33.3 % as medium, and 25 % as high. Nevertheless, there was no difference (p > 0.05) in the hydrophobicity percentage for the strains 16, 17, 19, 21, 25, 26, 28, and 29. 3.4. Exopolysaccharide production phenotype Fig. 2 (A-F) illustrates the production of EPS by LAB strains. P. pentosaceus (16), P. acidilactici (18), E. faecium (21), W. viridescens (25), L. casei (28) and L. fermentum (29) exhibited the phenotype “ropy”, which was observed as a thin filament when a colony was picked up with a bacteriological loop. 3.5. Antibacterial activity against foodborne pathogens The antibacterial activity results are provided in Fig. 3 (A-D). All LAB strains showed antibacterial activity against gram-negative pathogens. P. pentosaceus (16), E. faecium (21), L. mesenteroides subsp. mesenteroides (26) and L. casei (28), were classified as having a high inhibitory capacity (4.0–4.25 mm) against E. coli without statistical differences (p > 0.05). Antibacterial activity against Gram- positive pathogens was variable, most notably for L. monocytogenes, in which 50 % of the strains presented inhibition, with inhibition zones ranging from 3 to 4 mm, while 10 strains inhibited S. aureus. P. pentosaceus showed the highest antibacterial activity against all pathogens studied (>4 mm) but there was no significant difference (p > 0.05) with the strains 18, 21, 26, 28, and 29. 3.6. PCA analysis The results on the integration of probiotic properties as assessed with a PCA, including the tests of antibacterial activity, pH, and bile salt tolerance, autoaggregation, and hydrophobicity are presented in Fig. 4. This analysis showed two components, PC1 and PC2, explaining the greatest amount of variance for the probiotic properties data, with a final contribution of 44.07 % and 23.64 %, respectively, totaling 67.61 % of the variation. The PCA showed that antibacterial activity against all pathogens and autoaggregation were correlated with PC1; 1.0 % (w/v) bile salt and pH 3.0 tolerance, and hydrophobicity with chloroform were correlated with PC2 (Table 4). This suggests that the variables that were correlated to PC1 and PC2 contribute to the selection of the most suitable strains with probiotic properties. According to this, the strains that were grouped in the first quadrant (16, 21, 26, 28 and 29) were the probiotic candidates with the most potential. 4. Discussion In the selection of microorganisms with probiotic properties, a sequence of multiple tests is required to progressively reduce the number of probiotic candidates, according to the qualities exhibited, the industrial requirements, and the beneficial effects. The strains Fig. 3. Antibacterial activity of LAB strains against A. Salmonella Typhimurium ATCC 14028, B. Escherichia coli ATCC 25922, C. Staphylococcus aureus ATCC 25923 and D. Listeria monocytogenes ATCC 19111. S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 7 evaluated must comply with the in vitro evaluation guidelines proposed by the FAO/WHO [14,15]. One of the first probiotic tests is the safety evaluation, which guarantees no risk to human health. The studied strains did not have hemolysin or toxic by-products that could lyse red blood cells. These results agree with previous reports carried out for the genera Pediococcus [36], Leuconostoc [37], Enterococcus [38], Weissella [39], Lactococcus [40], and Lactobacillus [41], which is a requirement to classify bacteria as having Fig. 4. Principal Component Analysis (PCA) based on the probiotic properties antibacterial activity, pH, and bile salt tolerance, autoaggregation and hydrophobicity. A. Strain distribution, B. Distribution of probiotic properties. S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 8 Qualified Presumption of Safety (QPS) according to EFSA [42]. Another criterion that should be considered in the evaluation of a probiotic is sensitivity to antibiotics. In this study, all the strains belonging to the genera Pediococcus, Limosilactobacillus, and Lacticaseibacillus were sensitive to the antibiotics tested (Table 2), as stated by the EFSA [42], indicating that these genera do not present atypical resistance to clinically relevant antimicrobials. Likewise, other studies have reported the same sensitivity profile for these genera [43–45]. Erythromycin resistance in the strains Leuconostoc (17, 19 and 26), Weissella viridescens, and Lactococcus has been associated with the gene erm(B) [46–48] and in the case of Weissella viridescens, the gene catA has been associated with chloramphenicol resistance [49]. On the other hand, for some strains of Enterococcus there have been reports that identify them as multiresistant [38,50]. Most LAB are recognized as Generally Recognized As Safe (GRAS) or Qualified Presumption of Safety (QPS), however, there is a possibility that these strains could become reservoirs for resistance genes [51,52]. The evaluation of sensitivity to antibiotics of each strain was phenotypically done. The microdilution broth method is one of the most used phenotypic methods to detect antibiotic resistance. Therefore, the type of resistance that a strain may have (intrinsic or acquired) is unknown and must be verified by genotypic methods before ruling out potential candidate strains for probiotics [53]. The ability to survive the conditions of the gastrointestinal tract is one of the most important functional features to consider in the selection of a probiotic [54]. In this study, the pH of 6.5, 3.0, and 8.0 corresponding to the oral cavity, stomach, and large intestine, respectively, and bile salts in the small intestine were evaluated (0.3 % and 0.6 % (w/v)) [55], conditions that were tolerated by all strains. Several researchers have reported tolerance to these pH conditions for these species [44,56,57]. The different methods each strain employs to tolerate pH and bile salts, which are unique to each strain, may be the cause of the variance in the survival of the strains in the environment of the gastrointestinal system [38]. For example, Lactobacillus presents mechanisms for resistance to acidity such as proton pumps, the ability to alkalinize the external environment, modification of the lipid content of the membrane, and the formation of biofilms, which facilitate auto-aggregation and protection from extreme pH conditions [58,59]. The tolerance of pro biotics to bile salts is related to the enzyme Bile Salt Hydrolase (SBH), the active efflux of salts and bile acids, and the alteration of the membrane and cell wall [60,61]. Autoaggregation is a desired characteristic for probiotics as it ensures that they arrive at a high density in the intestine, protecting them from stress conditions [62] to enable their interaction with the host, contribute to the adhesion capacity and colonization of the GIT [63] and exert beneficial effects such as the inhibition of potential pathogens [33]. According to Cisneros [33], autoaggregation percentages equal to or lower than 10 % correspond to strains that are unable to autoaggregate, while strains with percentages higher than 10 % can naturally autoaggregate. In this study, 50 % of the strains presented autoaggregation values between 20 % and 35 %, as reported by several authors for the same species [64,65]. Different molecules associated with the property of auto-aggregation have been described, such as the aggregation promoting fact-related surface protein and exopolysaccharides in Pediococcus [66], the Promoting Factors of Aggregation (APF) in Lactobacillus [67], the clumping properties of Leuconostoc [68] and the enterococcal surface protein, aggregation substance, and collagen-binding protein for Enterococcus [69]. Hydrophobicity in probiotics has been related to the interaction between the bacterial surface and the epithelial cells of the in testine. It depends on non-polar molecules such as glycoproteins and polysaccharides present in the membranes and the cell wall [70]. This property is an indirect estimate of the successful adhesion and colonization of probiotics [71]. Therefore, its evaluation allows to characterize the capacity of the surface of the strains to interact with the mucosa and the cells of the intestine, but it is not a prerequisite for a strong adhesion [72]. The strains 19, 26, and 29 showed a higher affinity for the non-polar acid solvent (Chloroform) and a lower affinity for the ethyl acetate (non-polar basic solvent), coinciding with the reports of other authors [73,74], which indicates that these strains are electron donors because chloroform has the characteristics of an electron acceptor [75]. Visual screening of colonies in a solid culture medium is one of the most frequently used methods for the detection of the exo polysaccharide production phenotype. EPS production depends on the carbon and nitrogen sources, with sucrose being the sugar that promotes growth in screening EPS-producing colonies [76]. In this study, the strains of the genera Pediococcus, Limosilactobacillus, Lacticaseibacillus, E. faecium, and W. viridescens presented a ropy phenotype, indicating that they can produce exopolysaccharides. Other authors have reported that these species produce ropy colonies on sucrose-containing media [77,78]. There are no reports of EPS production by W. viridescens, but other species have been considered producers, including W. confusa, W. cibaria, and W. hellenica [79, 80], which means that the production of EPS is a strain-specific characteristic and not of an entire bacterial genus. Probiotics produce EPS on the cell envelope as capsules or paracrystalline layers to protect against phagocytosis, fag attack, desiccation, and osmotic stress, and have therapeutic applications with antimicrobial, immunomodulatory, anti-inflammatory, antioxidant, antitumor, anti viral, and cholesterol-lowering properties [81]. Table 4 Principal component coefficient matrix. Probiotic properties PC1 PC2 Antibacterial activity against E. coli 0.222 0.158 Antibacterial activity against S. Typhimurium 0.218 0.001 Antibacterial activity against S. aureus 0.193 0.018 Antibacterial activity against L. monocytogenes 0.235 0.126 Autoaggregation 0.118 − 0.268 Hidrophobicity with ethyl acetate − 0.064 − 0.222 Hidrophobicity with chloroform 0.050 0.264 Tolerance to pH 3.0 − 0.046 0.437 Tolerance to bile salts 1.0 % (w/v) − 0.200 0.218 S. Roldán-Pérez et al. Heliyon 9 (2023) e21558 9 Antibacterial activity is an essential factor in selecting probiotic bacteria due to their activity against foodborne pathogens. The results revealed that the cell-free culture supernatant (CFS) from strains 16, 18, 21, 26, 28 and 29 showed an inhibitory effect against all pathogens. Their activity against Gram-negative indicator bacteria was higher than that against Gram-positive bacteria, providing evidence that P. pentosaceus (16) had a higher inhibition (>4.0 mm) against all evaluated pathogens. These results coincide with reports made by other authors in these species [77,82]. This inhibitory activity of LAB has been attributed to its ability to produce different metabolites such as bacteriocin, organic acids (lactic, acetic, and propionic), ethanol, diacetyl and hydrogen peroxide [83]. 5. Conclusions Overall, Pediococcus, Limosilactobacillus and Lacticaseibacillus strains exhibited strong probiotic properties, such as being safe, having good tolerance to pH and bile conditions, autoaggregation, and hydrophobicity, showing a broad spectrum of antibacterial activities against foodborne pathogens such as L. monocytogenes and S. Typhimurium, and having an EPS ropy phenotype, for which they can be proposed as potential probiotics for studies in development on an industrial scale and their future application to elaborate functional food. Although, strains L. mesenteroides (19) and E. faecium (21) presented positive results in most of the probiotic tests carried out, they were resistant to a few clinically effective antibiotics; therefore, it is necessary to carry out more safety studies for these strains for their potential use as probiotics. The test carried out in this study constitute a preliminary evaluation of the possible probiotic use of these strains for the formulation of functional foods. However, technological tests such as studies on cell lines and cytotoxicity, as well as viability tests on food, need to be performed. Funding sources The authors are grateful to the Institución Universitaria Colegio Mayor de Antioquia and Universidad Nacional de Colombia sede Medellín for funding this work. Data availability statement Data included in article/supplementary material/referenced in article. CRediT authorship contribution statement Samantha Roldán Pérez: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Conceptualization. Sara Lucía Gómez Rodríguez: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Conceptualization. José Uriel Sepúlveda: Writing – review & editing, Supervision, Project adminis tration, Funding acquisition. Orlando Simón Ruiz Villadiego: Writing – review & editing, Supervision, Project administration, Funding acquisition. María Elena Márquez Fernández: Writing – review & editing, Supervision, Project administration, Funding acquisition. Olga Inés Montoya Campuzano: Writing – review & editing, Supervision, Project administration, Funding acquisition. Mónica María Durango Zuleta: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investi gation, Funding acquisition, Conceptualization. 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Tolerance to pH and bile salts 3.3 Autoaggregation and cell surface hydrophobicity 3.4 Exopolysaccharide production phenotype 3.5 Antibacterial activity against foodborne pathogens 3.6 PCA analysis 4 Discussion 5 Conclusions Funding sources Data availability statement CRediT authorship contribution statement Declaration of competing interest References