Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates

Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates Carolina Iraporda, Irene A. Rubel, Guillermo D. Manrique, Analía G. Abraham This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3976150/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The search for probiotic candidates is an area that accompanies the world trend of development of novel probiotic strains and new products. In recent years, unconventional sources of potential probiotic bacteria have been studied. Furthermore, nowadays there has been a growing interest in non-dairy probiotic products and fermented plant-based foods, which has led to the development of probiotic foods currently being presented as a research priority for the food industry. The aim of this work was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers. The results proved that the selected isolated LAB strains exhibited a high survival rate in the simulated gastrointestinal treatment, with non-hemolytic nor DNAse activity and antibiotic sensitivity. The isolated strains also showed antimicrobial activity against pathogen microorganisms, due to their acidification capacity. The molecular identification of the bacilli strains showed a high similarity with the genus Lentilactobacillus and, within this genus, with the species kosonis and curieae . Hence, these strains revealed potential probiotic in vitro characteristics that position them to be used in plant-based functional food. Jerusalem artichoke (Helianthus tuberosus L.) tubers lactic acid bacteria molecular identification probiotic potential safety Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Probiotic microorganisms are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al., 2014 ). The scientific community has grown increasingly the interest in probiotic research over the years due to the wide range of health benefits they provide for people and consumer demand. In this sense, the search for probiotic candidates is an area that accompanies the global trend of development of novel probiotic strains for their application in new products. Most of the microorganisms used as probiotics include bacteria and yeast. The microorganisms that have been traditionally considered as probiotics are those that belongs to the family Lactobacillaceae or the genus Streptococcus , Leuconostoc , Pediococcus , Propionibacterium , Enterococcus , Bifidobacterium , Bacillus or yeast such as Saccharomyces boulardii, Saccharomyces cerevisiae , Candida pintolopesii , Aspergillus niger and A. oryzae (Fuller & Fuller, 1992 ). Lactic acid bacteria (LAB) belonging to the genus formerly called Lactobacillus and Bifidobacterium are considered a major group of probiotic bacteria commonly used for both animals and human nutrition (Nousiainen et al., 2004 ; Soccol et al., 2010 ). It should be noted that since 2020, the Lactobacillus genus has been reclassified into more than 20 new genera (Zheng et al., 2020 ). These microorganisms are commonly isolated from traditional fermented foods and dairy products. Much research suggests that LAB strains from animal and human origin may have a good survival against gastric and intestinal stress factors; making them promising probiotic candidates (Panwar et al., 2021 ; Thakur et al., 2016 ). The high carbohydrate content, nutrient availability and the acidic environment of fruits and vegetables may favor the growth of LAB. In recent years, unconventional sources of potential probiotic bacteria have been studied, including some of non-intestinal origin and from non-dairy products, such as traditional fermented foods, grains (Pedersen et al., 2004 ; Sáez, Saavedra, Hebert, & Zárate, 2018 ), honey-comb (Tajabadi et al., 2013 ), air and soil (Chen et al., 2005 ; Yanagida, Chen, Shinohara, 2006 ; Zielińska & Kołożyn-Krajewska, 2018 ), and vegetables (Cele et al., 2022 ; Naeem et al., 2012 ; Patel et al., 2014 ; Somashekaraiah et al., 2019 ). Furthermore, nowadays there has been a growing interest in non-dairy probiotic products and fermented plant-based foods, which has led to the development of probiotic foods currently being presented as a research priority for the food industry (Betoret et al., 2012 ). The isolation, identification, and evaluation of safety and probiotic properties of ´new´ or ´wild´ strains of microorganisms from different sources require a systematic approach consisting of sequential evaluations to select candidate strain. The methodology and criteria used to evaluate probiotic candidates include assessing their ability to tolerate stressful conditions exerted by the human body, ability to interact with host epithelial cells, safety attributes (such as β-hemolysis, gelatinase, and DNAse enzyme activities), and sensitivity to antibiotics, antimicrobial activity, and competition with pathogens (Choudhary et al., 2019 ; Falah et al., 2019 ; Gharbi et al., 2019 ; Wang et al., 2018 ). Jerusalem artichoke ( Helianthus tuberosus L.) is an annual plant native from North America that is characterized by its high adaptability and resistance to adverse environmental conditions. Jerusalem artichoke tubers (JAT) store inulin as carbohydrate reserve, reaching up to 20% of its fresh weight (Kays & Nottingham, 2008; Rubel et al., 2021 ). The plant rhizosphere is an interesting environmental niche enriched with root secretions and various plant-associated bacteria and fungi. Thus, it can be a good source for isolating probiotics exhibiting competence for survival in the microbe-rich human gastrointestinal tract (Singhal et al., 2021 ). As suggested by Chen et al. ( 2005 ) the rhizospheres of fruit trees are good sources of LAB. Many traditional preparations are made from vegetables that are fermented by the bacteria that naturally exist on the raw material (Wacher et al., 2010 ). Moreover, adding LAB as starter cultures plays an important role in food fermentation, avoiding variations and inconstant properties of the naturally fermented final products, and reaching desirable organoleptic properties and preservative effect. Naturally fermented Jerusalem artichoke tubers added or not with LAB as starters showed that Weissella soli and Lactococcus lactis were the predominant group of LAB in the microbial community of the brine (Yokoi et al., 2006 ). The aim of this work was to evaluate the probiotic potential of LAB isolated from an unconventional source such as the Jerusalem artichoke tubers. The isolated LAB strains were characterized by in vitro tests for their biochemical and probiotic properties (acid and bile tolerance, survival after simulated gastrointestinal treatment, inhibition of pathogen microorganisms) and safety aspects (antibiotic susceptibility, hemolytic and DNAse activity) promoting their use for the development of vegetal-fermented food or other potential technological applications. Materials and methods Isolation of bacterial strains from Jerusalem artichoke tubers Lactic acid bacteria were isolated by culture enrichment, according to Endo et al. ( 2009 ), with minor modifications. Fresh Jerusalem artichoke tubers (JAT) were harvested and washed with sterile water. Then, small pieces of JAT were incubated aerobically at 37°C for 72 h, in a formulated medium containing (g/L): yeast extract (10), polypeptone (5), sodium acetate (2), tween 80 (0.5), MgSO 4 .7H 2 O (0.2), MnSO 4 .4H 2 O (0.01), FeSO 4 (0.01), NaCl (0.01), cycloheximide (0.05) with fructose (FYP) or inulin (IYP) as carbon source (10). After incubation, 100 µL of the enriched cultures were inoculated into fresh FYP or IYP broth and further incubated at 37°C until visible growth detection. Subsequently, serial dilutions of the cultures were plated onto FYP or IYP agar containing 5 g/L of CaCO 3 . All plates were incubated aerobically at 37°C. Colonies, randomly selected according to morphological differences (colony size and shape), were picked and purified by streaking on the suitable agar media and further characterized. Overnight cultures of the isolates were preliminarily assayed for Gram staining, microscopic morphology, catalase and oxidase activity. Gram-positive, catalase and oxidase-negative cocci and rods were selected, and stored in MRS broth containing skim milk (1:1) at -20°C. Genotypic characterization of selected bacteria Genomic DNA used as template was extracted according to Ruiz Rodríguez et al. ( 2016 ). Oligonucleotide primers 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3' were used to amplify the 16S ribosomal RNA gene. The obtained fragment of each isolate was purified and sequenced by the Sequencing Service of CCT-CONICET- Tucuman (San Miguel de Tucumán, Argentina). The sequences were analyzed using BLAST (The Basic Local Alignment Search Tool) from NCBI ( http://www.ncbi.nlm.nih.gov/BLAST ). The percentage of similarity was calculated as the product of the query cover and the percentage identity obtained. Morphological and biochemical tests The LAB isolates were studied according to their morphological and biochemical characteristics. Cell morphology Bacterial cultures in stationary phase grown in MRS agar and broth were mounted on microscopic slides and examined under a light microscope using oil immersion objectives. Cell morphology and cell arrangements were observed. Growth at 45°C and at high NaCl concentrations The selected bacterial cultures were transferred into MRS broth and incubated for 5 days at 45°C and into MRS broth containing NaCl (4 and 6.5%w/v) for 5 days at 37°C. The final pH and optical density (OD) at 600 nm of the medium were measured to verify the growth of the strains under each condition, and the growth was registered as positive or negative. Gas production from glucose In order to determine the homofermentative or heterofermentative metabolism of the selected LAB, CO 2 production from glucose was determined in MRS broth containing inverted Durham tubes. The presence of gas in Durham tubes during 48 h of observation indicates CO 2 production from glucose. Sugar fermentation The ability to use different sugars, oligo, and polysaccharides or sugar alcohols, including D-glucose, D-fructose, D-xylose, lactose, sucrose, D-raffinose, fructooligosaccharides, inulin with low or high polymerization degree, sorbitol, and mannitol, were evaluated according to He et al. ( 2021 ). These carbohydrates were used as the primary carbon source in MRS broth without glucose and meat extract. The 1%v/v cell suspension (10 8 cells/mL) was inoculated into the medium containing 2%w/v carbohydrate. The OD at 600 nm and pH were recorded and compared with the growth obtained in MRS without carbon source after 48 h of incubation at 37°C. Proteolytic capacity The selected LAB strains grown in MRS broth at 37°C during 48 h were streaked in milk agar and incubated for 72 h at 37°C. The appearance of a clear zone around the bacterial colonies confirms the ability of the strains to hydrolyze caseins to peptides and amino acids (Olajuyigbe & Ajele, 2005 ). Cell surface: autoaggregation The autoaggregation assay was performed according to Del Re et al. ( 2000 ). Briefly, the selected LAB grown for 48 h at 37°C in MRS broth, were then centrifuged for 10 min at 10000 xg. Subsequently, cells were washed three times with phosphate saline buffer (PBS). Further, obtained cells were suspended in sterile PBS to obtain a 10 8 CFU/mL concentration. A suspension of 5 mL was vortexed for 10 s and incubated at room temperature for 24 h. After this time, the OD at 600 nm was measured. The percentage of autoaggregation was expressed as follows: Autoaggregation (%) = [(OD 1 − OD 2 )/OD 1 ] × 100 Where OD 1 and OD 2 are the optical densities at the initial time and after 24 h, respectively. All experiments were performed in triplicate. Resistance to gastrointestinal passage and antimicrobial properties of the strains Acid tolerance The acid tolerance of the isolated strains was evaluated by inoculating active cultures in in MRS broth with pH adjusted to 2.5 with HCl 1N. Cell viable counts were done initially and followed by incubation at 37°C for 1.5 and 3 h. At each time, decimal dilutions were plated in MRS agar to determine the colony-forming units per mL (CFU/mL). The survival percentage was calculated as the relation between the final Log CFU/mL and the initial Log CFU/mL. Bile tolerance The bile tolerance of the selected LAB strains was evaluated by incubating bacterial culture in MRS broth containing 0.3%w/v bile salts (Sigma-Aldrich, USA) at 37°C for 24 h. An initial and final viable count was done in MRS agar. The survival percentage was calculated as the relationship between the final Log CFU/mL with respect to the initial Log CFU/mL. Tolerance to simulated gastrointestinal conditions The selected LAB strains grown in MRS at 37°C for 48 h were harvested and resuspended in a simulated gastric juice (NaCl 125 mM, KCl 7 mM, NaHCO 3 45 mM, pepsin 3 g/L, pH adjusted to 2.5) at OD 600 nm 0.5 (10 8 CFU/mL). The suspensions were incubated at 37°C for 1.5 h and then centrifuged 10 min 10000 x g . The bacterial pellet was then resuspended in simulated intestinal fluid (NaCl 22 mM, KCl 3.2 mM, NaHCO 3 7.6 mM, pancreatin 0.1%w/v, bovine bile salts 0.15%w/v, final pH adjusted to 8.0) and incubated at 37°C for 2.5 h. After treatment, samples were diluted in saline solution and plated on MRS agar to determine bacterial viability (Grimoud et al., 2010 ). The percentage of survival of the strains was calculated as the relation between Log CFU/mL after and before the sequential gastrointestinal treatment. Antimicrobial activity Double layer agar method The antimicrobial activity of the selected LAB strains against the foodborne pathogens Escherichia coli ATCC 11229 and Bacillus cereus ATCC 10876 was determined using the double-layer agar method, as described by Ripamonti et al. ( 2011 ). Overnight test cultures were spotted (5 µL) on the surface of MRS agar and incubated for 24 h at 37°C, and two different assays were carried out. In the first one, the LAB colonies grown in the surface of MRS agar plates were inactivated with chloroform for 30 min. Then 10 mL of brain heart infusion (BHI, Britania, Argentina) soft agar (0.7%w/v) inoculated with 50 µL of an overnight culture of each pathogen indicator grown in BHI was poured onto MRS agar plates. In the second one, the LAB colonies were not inactivated. The plates with double-layer agar were incubated aerobically at 37°C for another 24 h and the inhibition zones were subsequently measured (cm). Diffusion agar assay Evaluation of the antagonic activity of the LAB culture supernatants was screened by the agar well diffusion assay reported by Tejero-Sariñena et al., ( 2012 ) with some modifications. Briefly, Petri dishes were overlaid with 20 mL of BHI agar previously inoculated with 100 µL of an overnight culture of the foodborne pathogen indicator microorganisms grown in BHI broth ( E. coli or B. cereus ). Subsequently, cell-free supernatant, sterilized by filtration with 0.22 µm pore size membrane (Gamafil, Argentine), were placed into agar wells (40 µL). Also, cell-free supernatants neutralized with NaOH 3N and 20-fold concentrated by lyophilization were analyzed. The plates were incubated aerobically for 24 h at 37°C and the inhibition zones were subsequently measured (cm). Safety evaluation of selected LAB strains Antibiotic susceptibility The following antibiotics were tested at the recommended minimal concentration (mg/L) for homofermentative/heterofermentative Lactobacillus strains: ampicillin (1/4), gentamicin (16/16), streptomycin (16/64), erythromycin (1/1), clindamycin (1/1), tetracycline (4/8) and chloramphenicol (4/4) (EFSA FEEDAP Panel, 2012). All antibiotics were dissolved in the appropriate diluent for preparing concentrated stock solutions (10X). Then, stock solutions were diluted 1/5 in LSM broth (90% Iso-Sensitest plus 10% MRS). Bacterial cultures growth 48 h at 37°C were diluted 1:50 in LSM broth for inoculation of microdilution plates. Then, 100 µL of diluted inoculum in LSM were added to each well containing 100 µL of an antibiotic solution in LSM (2X). After incubating plates under aerobic conditions at 37°C for 48 h, OD at 600 nm was measured. The susceptibility of LAB strains was categorized as resistant (R) when the growth was higher than 10% concerning the control media without antibiotic, moderately sensitive (MS) when the growth was lower than 10%, or susceptible (S) when no growth was observed. Hemolytic activity The selected LAB cultures were streaked on blood agar (Britania, Argentine) and incubated for 72 h at 37°C under aerobic conditions. Green zones around the colonies suggested α-hemolysis, clear zones around the bacterial colonies indicated the presence of β-hemolysis, whereas no clear zones were recorded as non-hemolytic. DNAse activity The selected LAB isolates were streaked onto a deoxyribonuclease (DNAse) agar medium (Britania, Argentine) to test for the production of the DNAse enzyme. The plates were incubated at 37°C for 48 h under aerobic conditions and observed for the zone of DNAse activity after the addition of HCl 1N. A clear zone around the colonies was considered as positive (+) DNAse activity, whereas no clear zones were recorded as negative (-) (Shuhadha et al., 2017 ). Results Isolated bacterial strains After seeding FYP CaCO 3 agar plates with serial dilutions of the enriched medium, many colonies surrounded by a clear zone due to the local solubilization of CaCO 3 by presumptive lactic acid bacteria from the rhizosphere of the tubers, were obtained. Forty-eight colonies were isolated, 35 cocci and 13 bacilli, Gram positive, catalase and oxidase negative. Considering the acidification power in FYP broth, 3 cocci (named as A, E, J) and 3 rods (named as F, G, H) were selected for their further characterization and assays of their probiotic capacity as a preliminary step for their potential use in the development of vegetal-fermented food. Neither of the selected isolated strains were able to grow at 45°C nor with NaCl 4,5 or 6%w/v. The 3 strains with coccus morphology did not produced gas from glucose, while the 3 rod-shaped strains produced gas from glucose, indicating homofermentative and heterofermentative metabolism, respectively. The colony macroscopic aspect in MRS agar and microscopic morphology subjected to a Gram stain of the selected strains grown at 37°C are shown in Fig. 1 . A difference in the bacterial morphology in liquid and solid medium was observed for the 3 selected rod-shaped strains. They presented a typical bacilli shape in the broth, while in solid medium they grew forming shorter bacilli cells. Meanwhile, the coccus-shaped strain presented the same bacterial morphology when they grew in MRS broth or solid media. All the strain grew in MRS broth at 37°C, the pH decreased during bacterial growth, and they reached the stationary phase in 24 h with concentrations around 9 Log CFU/mL (Figs. 2 and 3 ). The acidification in the MRS broth of the cocci was slightly higher than that of the bacilli, reaching final pH values of 3.6–3.8 and 4.1–4.3 after 48 h, respectively (Figs. 2 and 3 ). The three rod-shaped strains were able to ferment glucose and fructose, while the three coccus-shaped strains, in addition to these carbohydrates, they also fermented sucrose and mannitol. The isolated strains revealed no considerable casein degradation during their aerobic growth in milk agar plates. The isolated strains A, E and J showed autoaggregation capacity after 24 h of 62.8 ± 1.5; 58.3 ± 4.8 and 71.3 ± 3.9% respectively; whereas the strains F, G and H presented values of 77.7 ± 9.7; 61.2 ± 11.4 and 81,5 ± 10.2%, respectively. In this instance the focus was placed in the rod-shaped strains. The amplification products corresponding to the 16S RNA gene gave an approximately 1400 bp amplicon. The analysis of the sequences obtained, revealed similarity of the strains F, G and H with isolates belonging to the Phylum ´Firmicutes´, class ´ Bacilli´ , order ´ Lactobacillales´ , family ´ Lactobacillaceae ´, genus ´ Lentilactobacillus ´. Within this genus, both strains F and H, showed high similarity with strains belonging to the species Lentilactobacillus kosonis , with a percentage of similarity 94.2% (percentage of identity of 96.1%) and 96.4% (percentage of identity 97.3%), followed by Lentilactobacillus curieae percentage of similarity 92.3% (percentage of identity of 96.1%) and 91.8% (percentage of identity 95.6%), for the strains F and H, respectively. Meanwhile the percentage of similarity of the strain G with strains belonging to the species Lentilactobacillus kosonis and Lentilactobacillus curieae was 91.2% (percentage of identity 92.1%). Likewise, percentage of similarity higher than 90% were also obtained concerning other species from the genus Lentilactobacillus , such as Lentilactobacillus senioris and Lentilactobacillus fungorum for the strain G, and Lentilactobacillus sunkii , Lentilactobacillus otakiensis , Lentilactobacillus buchneri and Secundilactobacillus odoratitofui for the strain H. Probiotic properties All the strains survived in acidic conditions, in presence of bile salt, and after the simulated sequential gastrointestinal treatment (GIT) (Table 1 ). After 1.5 h of exposure at pH 2.5, the percentage of survival were higher than 94.8%, except for the strain J, which presented a significantly lower survival. Meanwhile, after 3 h, the rod-shaped strains presented a significantly higher percentage of survival than the coccus-shaped strains. After the bile salt exposure, the rod-shaped strains presented high resistance (> 94.2%) without significant differences in their percentage of survival. Among the isolated coccus, the strain A show survival percentage after bile salt treatment of 80.2%, with no significant differences with the strain J, and significantly higher than the strain E. A high resistance was also observed after the sequential simulated GIT challenge for all the selected strains. The rod-shaped strains presented significantly higher survival than the coccus-shaped strains. Table 1 Percentage of bacterial survival after acidic treatment (pH 2.5, 37°C, 1.5 h or 3 h), bile salt exposure (0.3%w/v, 37°C, 24 h) and simulated sequential gastrointestinal treatment. Strain Acid treatment 1.5 h Acid treatment 3 h Bile salt treatment GIT Coccus-shaped A 96.7 ± 1.4 B b 63.4 ± 4.1 A a 80.2 ± 2.0 AB b 74.1 ± 0.9 a A E 94.8 ± 0.6 B b 58.5 ± 1.6 A a 65.6 ± 8.4 A a 73.7 ± 2.6 a A J 83.2 ± 5.2 A a 76.6 ± 2.2 B b 75.0 ± 9.2 A ab 71.2 ± 2.1 a A Rod-shaped F 97.8 ± 1.44 B a 96.1 ± 0.9 C a 108.1 ± 8.8 C a 94.6 ± 1.1 b C G 96.8 ± 1.5 B a 95.8 ± 2.0 C a 108.5 ± 11.9 C a 93.2 ± 0.2 b BC H 97.2 ± 0.8 B a 96.4 ± 3.1 C a 94.2 ± 4.3 BC a 88.9 ± 2.6 a B GIT: Gastrointestinal treatment. Values are represented as mean ± SD of at least three independent replicates. Superscripts with different capital letters indicate significant differences between strains, for each column. Lowercase superscripts indicate significant differences between the cocci-shaped or rod-shaped strains, for each column. ANOVA followed by Tukey test (α = 0.05). Both viable cells and the metabolites of all the selected strains exerted an inhibitory effect on the pathogenic microorganisms E. coli and B. cereus . Moreover, this antimicrobial activity was significantly higher for the rod-shaped strains than that observed for the coccus-shaped strains. The agar plates with inhibition zones produced by the strains F, G, and H against E. coli and B. cereus are shown in Fig. 4 . The strain F exerted an inhibitory effect significantly higher (p < 0.05) against both pathogens than the rest of the strains, with diameters of inhibition of 3.0 ± 0.1 cm and 3.7 ± 0.1 for E. coli and B. cereus , respectively. The lowest inhibition was exerted by the strain A against B. cereus (diameter of inhibition 1.73 ± 0.1 cm) and strains A and J with E. coli (diameters of inhibition 2.1 ± 0.1 cm). The inhibition exerted by metabolites of rod-shaped strains against B. cereus did not show significant differences (diameters of inhibition between 4.1 and 4.4 cm). Finally, the inhibition against E. coli exerted by the rod-shaped strains produced diameters between 3.9 and 4.3 cm. On the other hand, the concentrated and neutralized culture supernatants of any of the selected strains show inhibitory effect against the pathogens analyzed (data not shown). Safety aspects None of the selected strains showed hemolytic capacity or DNAse activity. These attributes indicated safety aspects for these strains (Table 2 ). Moreover, the isolated strains were sensitive to the antibiotic ampicillin, streptomycin, erythromycin, clindamycin, gentamycin, tetracycline and chloramphenicol, at concentrations below the cut-off limit established by the EFSA ( 2012 ), for heterofermentative and homofermentative Lactobacillus strains and therefore they could be classified as sensitive (Table 2 ), however a moderate sensitivity was observed for the three rod-shaped strains against tetracycline. Table 2 Antibiotic sensibility, hemolytic and DNAse activity of the isolated strains. Strain AMP STR ERT CLI GEN TET CLO Hemolytic capacity DNAse activity Coccus-shaped A S S S S S S S - - E S S S S S S S - - J S S S S S S S - - Rod-shaped F S S S S S MS S - - G S S S S S MS S - - H S S S S S MS S - - S: sensitive. MS: moderately sensitive. R: Resistant. (-): Negative. (+): Positive. Discussion The rhizosphere is an interesting environmental niche enriched in plant secretions and soil-associated bacteria and fungi. Thus, it can be thought as a potential source for isolating competitive bacterial strains exhibiting probiotic properties in the human gastrointestinal tract (Singhal et al., 2021 ). The strains A, E and J resulted Gram positive coccus, catalase and oxidase negative, homofermentative, unable to growth at 45°C nor with NaCl 6%w/v, and gamma-hemolytic, so they could be classified as Streptococcus . The genus formerly called Lactobacillus displays a great level of genetic diversity. The reclassification of the formerly Lactobacillus genus in 23 new genera proposed in 2020 by Zheng et al. ( 2020 ) was considered for classifying the rod-shaped isolated strains. The molecular analysis of the strains F, G and H allowed them to be classified as members of the genus Lentilactobacillus . In recent years, strains with probiotic potential that were identified within the genus Lentilactobacillus have been isolated and from vegetable and fermented food sources (Carasi et al., 2022 ; Ebrahimi et al., 2017 ; Wu et al., 2021 ). The genus Lentilactobacillus is mainly represented by Gram-positive, rod-shaped, catalase negative, heterofermentative strains, that mostly grow at 15°C and some also grow at 45°C. Most of the strains that constitute this genus were isolated from silage, fermented vegetables, wine and cereal mashes (Zheng et al., 2020 ). All these sources present environmental characteristics that can be considered similar to those of the Jerusalem artichoke tubers. Moreover, strains in this genus lead a free-living lifestyle, and generally metabolize a broad spectrum of pentoses, hexoses, and disaccharides. The highest percentage of similarity found in this work was 96.4% for the isolate referred as H with the strain Lentilactobacillus kosonis strain C06.No73T, isolated from a traditional Japanese fermented beverage called kôso (Chiou et al., 2021 ), closely related also with the previously described Lentilactobacillus curieae CCTCC M 2011381T from stinky tofu (Lei et al., 2013 ). The similarity percentage of the isolated strains F and G were below 96%, being the highest 94.2% with Lentilactobacillus kosonis and 91.2% with Lentilactobacillus kosonis and Lentilactobacillus curieae , for F and G, respectively. Considering that a percentage of similarity higher than 90% was observed concerning several species, such as Lentilactobacillus senioris , L. fungorum , L. sunkii , L. otakiensis , L. buchneri and Secundilactobacillus odoratitofui , with which they also share ecological and metabolic properties, however other molecular studies could contribute to specify the identification of the strains at the species level (Zheng et al., 2020 ). In the base of the 16S rRNA gene sequence Lei et al. ( 2013 ) showed that the strain L. cuerieae CCTCC M 2011381T was closely related to L. senioris JCM 17472T, similar results with the obtained in the present work for rod-shaped strains isolated form Jerusalem artichoke tubers. It is worth to mention that the strain L. curieae CCTCC M2011381 showed a great potential as starter culture in fermentation of plant foodstuff (Liu et al., 2019). In another hand, Lentilactobacillus buchneri subsp. buchneri, Levilactobacillus brevis , and Limosilactobacillus fermentum were isolated and identified from Jordanian traditional pickled and fermented foods (Abudoleh et al., 2021 ). The autoaggregation capacity can favor the bacterial adhesion to intestinal epithelial cells, allowing bacteria to form a barrier and prevent the adhesion of undesirable microorganisms (Saito et al., 2019 ). Therefore, a high autoaggregation capacity is desirable for potential probiotic candidates. As suggested by Kos et al. ( 2003 ) the autoaggregation capacity is related to the type of proteins on the cell surface. The percentages of autoagregation obtained for the strains isolated from Jerusalem artichoke tubers in this work were high (> 50%). Among the isolated strains, the highest autoaggregation capacity was observed for the strain H (81,5 ± 10.2%) followed by the strain F (77.7 ± 9.7%). In general, lower percentage of autoagreggation were reported for LAB strains isolated from different vegetable sources. Fonseca et al. ( 2021 ) reported that LAB strains isolated from food and fermented vegetable foods, presented percentage of autoaggregation from 16.5 to 52.6%. Various LAB strains isolated from ethnic pickled bamboo shoot products displayed values of autoaggregation between 20 and 55%, in particular the strains Lv. brevis FS2.1, P. pentosaceus FS36.2, Lb. argentoratensis FS40.1, and P. pentosaceus FS34.1, showed the highest values (Kanpiengjai et al., 2022 ). Also, percentage of autoaggregation ranging from 2 to 28%, were reported for LAB strains isolated from fermented grains of Chinese baijiu (Fan et al., 2022 ). The high autoggregation obtained for LAB strains isolated from the Jerusalem artichoke tubers represent a positive aspect for their survival in adverse conditions resulting from a lesser exposure and also favorable for their intestinal adhesion capacity. On another hand, the selected LAB strains isolated in this work were able to ferment only a few carbohydrates, and as expected, the strains do not show proteolytic capacity. The survival of strains after the passage through the gastrointestinal tract (GIT), high acidic and bile salt levels (Han et al., 2021 ; Urdaneta & Casadesús, 2017 ), as well as the adhesion to epithelial cells capacity (Garcia-Gonzalez et al., 2018 ; Petrova et al., 2019 ), are important preliminary tests to claim about potential probiotic properties of a microorganism (Krawczyk et al., 2021 ; Qi et al., 2021 ). Moreover, no scientific consensus exists on the pH and bile concentration to which probiotic strains should be tolerant (Zago et al., 2011 ). The survival rates after acidic, bile salts, and simulated gastrointestinal treatments for the isolated strains were high. This assay demonstrated the resistance of the strains against adverse environments and their potential ability to reach the colon in high concentrations when consumed as part of food. Similar results were reported by other authors for LAB strains isolated from different natural sources (Delgado et al., 2007 ; Ramos et al., 2013 ; Vinderola et al., 2008 ; Zago et al., 2011 ). In particular, Singhal et al. ( 2021 ) reported that Lactiplantibaciullus plantarum strains isolated from rhizospheric soil remained viable in the presence lysozyme, after simulated gastric juice, and moderate bile salts exposure. Also, the strain Leuconostoc mesenteroides subsp. mesenteroides isolated from fermented carrot ang ginger brine exhibited high tolerance to acid, bile, and lysozyme (Cele et al., 2022 ). Abudoleh et al. ( 2021 ) reported only 1–2 Log CFU/mL reduction after simulated GIT for LAB strains isolated from plant-based fermented foods. Also, LAB strains isolated from a naturally fermenting coconut palm nectar could resist the simulated GIT without losing viability (Somashekaraiah et al., 2019 ). In line, many LAB strains isolated from Neera, resisted to simulated GIT and presented good antimicrobial activity (Somashekaraiah et al., 2019 ). The selected LAB strains of the present work showed a remarkable antimicrobial activity against the pathogenic bacteria E. coli and B. cereus , without producing bacteriocin-like substances, like most of the probiotic candidates isolated from unconventional sources. The inhibition results were attributed to the effect of the organic acids produced during bacterial growth, since no inhibition was exerted by the concentrated and neutralized culture supernatants, suggesting that they did not produce bacteriocin-like substances. In concordance, Fhoula et al. ( 2013 ) reported that culture supernatants of LAB isolated from rhizosphere exhibited strong inhibitory activity against several pathogenic bacteria, but only a few neutralized supernatants showed antagonistic activity against the tested pathogens. Bacterial strains isolated from traditional fermented Indian food, identified as Lactiplantibacillus plantarum 86 and Weissella cibaria 92, showed considerable antimicrobial activity against Gram-positive and Gram-negative pathogens (Patel et al., 2014 ). In contrast, Min Hsiu et al. ( 2016 ) informed that beneficial LAB isolated from fresh fruits and vegetables produced bacteriocin and other antimicrobial substances active against pathogenic bacteria and fungi. Safety aspects are essential when selecting probiotic candidates to support their potential use in food applications. In the present work, the selected LAB strains were sensitive to antibiotics for human and animal use, complying with European regulated cut-off values. Other authors reported that LAB isolates from Neera were resistant to some of the antibiotics tested; however, it is accepted that it is rarely their risk of infection outside healthcare situations (Sanders et al., 2010 ). Moreover, Arias and Murray, ( 2012 ) and Hanchi et al. ( 2018 ) reported that plant-origin strains generally display low virulence levels. It is important to remark that before using new isolates in food or feed formulations, their virulence and antimicrobial resistance genes must be verified to prevent the horizontal gene transfer for antibiotic resistance (EFSA FEEDAP Panel, 2018). Probiotic properties, safety aspects, and beneficial health effect must be reinforced by other in vitro and in vivo studies, if strains are intended to be used for food applications. Finally, this research confirms that the plant rhizosphere might be explored as a valuable ecological niche representing a source of strains with essential and desirable probiotic potential, that promote their use in plant-based foods. Conclusion In summary, in this work, lactic acid bacteria were isolated from an unexplored source, such as the Jerusalem artichoke tubers, for the first time, and results showed that they were a reservoir of promising probiotic candidates. The results proved that the selected isolated LAB exhibited a high survival rate in the simulated gastrointestinal treatment, with non-hemolytic activity and antibiotic sensitivity. The isolated strains also showed antimicrobial activity against pathogen microorganisms, mainly attributed to their acidification capacity. The molecular identification of the bacilli strains showed a high similarity with the genus Lentilactobacillus and, within this genus, with the species kosonis and curieae . Hence, these strains revealed potential probiotic in vitro characteristics that position them to be used in plant-based functional food. The promising results promote further studies on the in vivo probiotic potential. Declarations Acknowledgements CI, IR and AGA are researcher from the National Council of Scientific and Technological Research (CONICET, Argentina) and GDM is researcher from de Commission of Scientific Investigations (CIC, Argentina). Authors are grateful for the financial support received from the research project PICT2019-0211 of the National Agency for Promotion of Scientific and Technical Research (ANPyCT-FONCyT, Argentina). Funding This work was supported by the research project PICT2019-0211 of the National Agency for Promotion of Scientific and Technical Research (ANPyCT-FONCyT, Argentina). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by C. Iraporda and I.A. Rubel. The first draft of the manuscript was written by C. Iraporda and I.A. Rubel all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval This work did not require ethics approval. References Abudoleh, S.M., Hamdan, S.O., Mahasneh, A.M., Al-Khani, Z.M., & Talhouni, A.A. (2021). Isolation and characterization of potential probiotic bacteria from jordanian traditional pickled and fermented foods. Acta Poloniae Pharmaceutica , 78(4), 515-520. DOI: 10.32383/appdr/141300 Arias, C.A. & Murray, B.E. (2012). The rise of the Enterococcus: beyond vancomycin resistance. Nat. Rev. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3976150","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274386150,"identity":"009f8af2-ec37-4aab-9e4c-14428f5ac710","order_by":0,"name":"Carolina Iraporda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie2Ru0rEQBSGTxiYNLOmPbDkHQYC2+RlMiwknS5sk8JibGbLtLFxX0HfIHIgNrFXAhIRtg4INoKY6EYUxktpMV8zw8A3/7kAOBz/EfZ+eNo/6arxxr+8/6gIkn9UYFJwKS1fWQg2s/vHVX4XFqcPXtUDHR74VHeQx0r7151NQfKjedmso7JdwmUJtOYizSQ0mdIikzZFMg7zmUmUbo8qEkDKoFigZ0hpSK2FDQp7HpXtLcFeCZ7QexmUYPedwt9Szm/YpAiOnh4UtKcgcR6XTRJdNGMvMlNGpAtM6iwyaE8Jipq1qzwJz66I9f0wqO2Gdtgfx2ER2FNGPq1gGlECHzv9TXE4HA6HhVd9R1nZI8GnKQAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Provincia de Buenos Aires","correspondingAuthor":true,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Iraporda","suffix":""},{"id":274386151,"identity":"c0d66ac7-717d-49f7-a178-40cc19969c94","order_by":1,"name":"Irene A. Rubel","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Provincia de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"A.","lastName":"Rubel","suffix":""},{"id":274386152,"identity":"00957d7b-aed0-4f3f-8239-64d13c8baf3d","order_by":2,"name":"Guillermo D. Manrique","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Provincia de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Guillermo","middleName":"D.","lastName":"Manrique","suffix":""},{"id":274386153,"identity":"995f1ce1-8ca9-4ac4-a62f-6df1acea88ba","order_by":3,"name":"Analía G. Abraham","email":"","orcid":"","institution":"Universidad Nacional de La Plata, Provincia de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Analía","middleName":"G.","lastName":"Abraham","suffix":""}],"badges":[],"createdAt":"2024-02-21 15:44:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3976150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3976150/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51633716,"identity":"366b0737-a57e-4d6a-b8f6-498cc7c2a3cd","added_by":"auto","created_at":"2024-02-26 10:21:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":965032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColony aspects and Gram stain of isolated selected strains\u003c/strong\u003e. \u003cstrong\u003e(A) \u003c/strong\u003eColony aspect in MRS agar 48 h 37 °C (left column), Gram stain of bacteria grown in MRS agar (middle column) and MRS broth, 48 h 37 °C (right column). Rod-shaped strains F (top row) G (middle row) and H (bottom row). \u003cstrong\u003e(B) \u003c/strong\u003eColony aspect in MRS agar 48 h 37 °C (left column), Gram stain of bacteria grown in MRS agar (middle column) and MRS broth, 48 h 37 °C (right column). Rod-shaped strains A (top row) E (middle row) and J (bottom row).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/728f0071cdd3e8b82ae0eaed.jpg"},{"id":51633718,"identity":"70547f9e-d839-4d02-8213-80942ed52fc0","added_by":"auto","created_at":"2024-02-26 10:21:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":673604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epH and bacterial growth of rod-shaped selected strain\u003c/strong\u003e \u003cstrong\u003ein\u003c/strong\u003e \u003cstrong\u003eMRS broth. \u003c/strong\u003eBacterial growth (CFU/mL) in MRS broth at 37 °C. \u003cstrong\u003e(B) \u003c/strong\u003epH of MRS broth during bacterial growth at 37 °C. (●) Strain F (■) Strain G and (▲) Strain H.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/4e3c1c314831314a57ea93f2.jpg"},{"id":51633717,"identity":"e2b93cd6-2eb0-4cf5-b2c4-3ce6ce56af26","added_by":"auto","created_at":"2024-02-26 10:21:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":666129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epH and bacterial growth of coccus-shaped selected strain\u003c/strong\u003e \u003cstrong\u003ein\u003c/strong\u003e \u003cstrong\u003eMRS broth. (A) \u003c/strong\u003eBacterial growth (CFU/mL) in MRS broth at 37 °C. \u003cstrong\u003e(B) \u003c/strong\u003epH of MRS broth during bacterial growth at 37 °C. (●) Strain A (■) Strain E and (▲) Strain J.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/59c77f13b99d766d7b1621b2.jpg"},{"id":51634085,"identity":"0e514433-e734-44d0-a569-33bad194083b","added_by":"auto","created_at":"2024-02-26 10:29:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2422261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of isolated strains and their metabolites. (A)\u003c/strong\u003e Inhibitory effect against \u003cem\u003eE. coli\u003c/em\u003e of metabolites (top row) and vegetative cells (bottom row) of the strain F (left column), G (middle column) and H (right column). \u003cstrong\u003e(B)\u003c/strong\u003e Inhibitory effect against \u003cem\u003eB. cereus\u003c/em\u003e of metabolites (top row) and vegetative cells (bottom row) of the strain F (left column), G (middle column) and H (right column).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/8d6a1829ab68a4fdb1c5649c.jpg"},{"id":64291721,"identity":"624255e9-e5b0-44cd-bf55-b1aebec8b205","added_by":"auto","created_at":"2024-09-11 09:44:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5603286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/b95dc717-7fd9-484d-b490-42f0170113ce.pdf"},{"id":51633720,"identity":"16ceb6c5-d0fb-4c62-afc0-6371ecfd8e25","added_by":"auto","created_at":"2024-02-26 10:21:29","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":135284,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorchecklistWJMB.docx","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/534e2c8d56021510e0fc1c53.docx"},{"id":51633721,"identity":"84630535-bc81-45a0-b414-0b5af992b41e","added_by":"auto","created_at":"2024-02-26 10:21:29","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":818167,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976150/v1/3e618788c23cd8789e9df447.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProbiotic microorganisms are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The scientific community has grown increasingly the interest in probiotic research over the years due to the wide range of health benefits they provide for people and consumer demand. In this sense, the search for probiotic candidates is an area that accompanies the global trend of development of novel probiotic strains for their application in new products. Most of the microorganisms used as probiotics include bacteria and yeast. The microorganisms that have been traditionally considered as probiotics are those that belongs to the family \u003cem\u003eLactobacillaceae\u003c/em\u003e or the genus \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eLeuconostoc\u003c/em\u003e, \u003cem\u003ePediococcus\u003c/em\u003e, \u003cem\u003ePropionibacterium\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e or yeast such as \u003cem\u003eSaccharomyces boulardii, Saccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003eCandida pintolopesii\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e (Fuller \u0026amp; Fuller, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Lactic acid bacteria (LAB) belonging to the genus formerly called \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e are considered a major group of probiotic bacteria commonly used for both animals and human nutrition (Nousiainen et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Soccol et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It should be noted that since 2020, the \u003cem\u003eLactobacillus\u003c/em\u003e genus has been reclassified into more than 20 new genera (Zheng et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These microorganisms are commonly isolated from traditional fermented foods and dairy products. Much research suggests that LAB strains from animal and human origin may have a good survival against gastric and intestinal stress factors; making them promising probiotic candidates (Panwar et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Thakur et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The high carbohydrate content, nutrient availability and the acidic environment of fruits and vegetables may favor the growth of LAB. In recent years, unconventional sources of potential probiotic bacteria have been studied, including some of non-intestinal origin and from non-dairy products, such as traditional fermented foods, grains (Pedersen et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; S\u0026aacute;ez, Saavedra, Hebert, \u0026amp; Z\u0026aacute;rate, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), honey-comb (Tajabadi et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), air and soil (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yanagida, Chen, Shinohara, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zielińska \u0026amp; Kołożyn-Krajewska, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and vegetables (Cele et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Naeem et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Patel et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Somashekaraiah et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, nowadays there has been a growing interest in non-dairy probiotic products and fermented plant-based foods, which has led to the development of probiotic foods currently being presented as a research priority for the food industry (Betoret et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The isolation, identification, and evaluation of safety and probiotic properties of \u0026acute;new\u0026acute; or \u0026acute;wild\u0026acute; strains of microorganisms from different sources require a systematic approach consisting of sequential evaluations to select candidate strain. The methodology and criteria used to evaluate probiotic candidates include assessing their ability to tolerate stressful conditions exerted by the human body, ability to interact with host epithelial cells, safety attributes (such as β-hemolysis, gelatinase, and DNAse enzyme activities), and sensitivity to antibiotics, antimicrobial activity, and competition with pathogens (Choudhary et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Falah et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gharbi et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eJerusalem artichoke (\u003cem\u003eHelianthus tuberosus\u003c/em\u003e L.) is an annual plant native from North America that is characterized by its high adaptability and resistance to adverse environmental conditions. Jerusalem artichoke tubers (JAT) store inulin as carbohydrate reserve, reaching up to 20% of its fresh weight (Kays \u0026amp; Nottingham, 2008; Rubel et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The plant rhizosphere is an interesting environmental niche enriched with root secretions and various plant-associated bacteria and fungi. Thus, it can be a good source for isolating probiotics exhibiting competence for survival in the microbe-rich human gastrointestinal tract (Singhal et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As suggested by Chen et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) the rhizospheres of fruit trees are good sources of LAB. Many traditional preparations are made from vegetables that are fermented by the bacteria that naturally exist on the raw material (Wacher et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, adding LAB as starter cultures plays an important role in food fermentation, avoiding variations and inconstant properties of the naturally fermented final products, and reaching desirable organoleptic properties and preservative effect. Naturally fermented Jerusalem artichoke tubers added or not with LAB as starters showed that \u003cem\u003eWeissella soli\u003c/em\u003e and \u003cem\u003eLactococcus lactis\u003c/em\u003e were the predominant group of LAB in the microbial community of the brine (Yokoi et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this work was to evaluate the probiotic potential of LAB isolated from an unconventional source such as the Jerusalem artichoke tubers. The isolated LAB strains were characterized by \u003cem\u003ein vitro\u003c/em\u003e tests for their biochemical and probiotic properties (acid and bile tolerance, survival after simulated gastrointestinal treatment, inhibition of pathogen microorganisms) and safety aspects (antibiotic susceptibility, hemolytic and DNAse activity) promoting their use for the development of vegetal-fermented food or other potential technological applications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of bacterial strains from Jerusalem artichoke tubers\u003c/h2\u003e \u003cp\u003eLactic acid bacteria were isolated by culture enrichment, according to Endo et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), with minor modifications. Fresh Jerusalem artichoke tubers (JAT) were harvested and washed with sterile water. Then, small pieces of JAT were incubated aerobically at 37\u0026deg;C for 72 h, in a formulated medium containing (g/L): yeast extract (10), polypeptone (5), sodium acetate (2), tween 80 (0.5), MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO (0.2), MnSO\u003csub\u003e4\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO (0.01), FeSO\u003csub\u003e4\u003c/sub\u003e (0.01), NaCl (0.01), cycloheximide (0.05) with fructose (FYP) or inulin (IYP) as carbon source (10). After incubation, 100 \u0026micro;L of the enriched cultures were inoculated into fresh FYP or IYP broth and further incubated at 37\u0026deg;C until visible growth detection. Subsequently, serial dilutions of the cultures were plated onto FYP or IYP agar containing 5 g/L of CaCO\u003csub\u003e3\u003c/sub\u003e. All plates were incubated aerobically at 37\u0026deg;C. Colonies, randomly selected according to morphological differences (colony size and shape), were picked and purified by streaking on the suitable agar media and further characterized. Overnight cultures of the isolates were preliminarily assayed for Gram staining, microscopic morphology, catalase and oxidase activity. Gram-positive, catalase and oxidase-negative cocci and rods were selected, and stored in MRS broth containing skim milk (1:1) at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenotypic characterization of selected bacteria\u003c/h2\u003e \u003cp\u003eGenomic DNA used as template was extracted according to Ruiz Rodr\u0026iacute;guez et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Oligonucleotide primers 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3' were used to amplify the 16S ribosomal RNA gene. The obtained fragment of each isolate was purified and sequenced by the Sequencing Service of CCT-CONICET- Tucuman (San Miguel de Tucum\u0026aacute;n, Argentina). The sequences were analyzed using BLAST (The Basic Local Alignment Search Tool) from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/BLAST\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/BLAST\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The percentage of similarity was calculated as the product of the query cover and the percentage identity obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMorphological and biochemical tests\u003c/h2\u003e \u003cp\u003eThe LAB isolates were studied according to their morphological and biochemical characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell morphology\u003c/h2\u003e \u003cp\u003eBacterial cultures in stationary phase grown in MRS agar and broth were mounted on microscopic slides and examined under a light microscope using oil immersion objectives. Cell morphology and cell arrangements were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGrowth at 45\u0026deg;C and at high NaCl concentrations\u003c/h2\u003e \u003cp\u003eThe selected bacterial cultures were transferred into MRS broth and incubated for 5 days at 45\u0026deg;C and into MRS broth containing NaCl (4 and 6.5%w/v) for 5 days at 37\u0026deg;C. The final pH and optical density (OD) at 600 nm of the medium were measured to verify the growth of the strains under each condition, and the growth was registered as positive or negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGas production from glucose\u003c/h2\u003e \u003cp\u003eIn order to determine the homofermentative or heterofermentative metabolism of the selected LAB, CO\u003csub\u003e2\u003c/sub\u003e production from glucose was determined in MRS broth containing inverted Durham tubes. The presence of gas in Durham tubes during 48 h of observation indicates CO\u003csub\u003e2\u003c/sub\u003e production from glucose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSugar fermentation\u003c/h2\u003e \u003cp\u003eThe ability to use different sugars, oligo, and polysaccharides or sugar alcohols, including D-glucose, D-fructose, D-xylose, lactose, sucrose, D-raffinose, fructooligosaccharides, inulin with low or high polymerization degree, sorbitol, and mannitol, were evaluated according to He et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These carbohydrates were used as the primary carbon source in MRS broth without glucose and meat extract. The 1%v/v cell suspension (10\u003csup\u003e8\u003c/sup\u003e cells/mL) was inoculated into the medium containing 2%w/v carbohydrate. The OD at 600 nm and pH were recorded and compared with the growth obtained in MRS without carbon source after 48 h of incubation at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eProteolytic capacity\u003c/h2\u003e \u003cp\u003eThe selected LAB strains grown in MRS broth at 37\u0026deg;C during 48 h were streaked in milk agar and incubated for 72 h at 37\u0026deg;C. The appearance of a clear zone around the bacterial colonies confirms the ability of the strains to hydrolyze caseins to peptides and amino acids (Olajuyigbe \u0026amp; Ajele, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell surface: autoaggregation\u003c/h2\u003e \u003cp\u003eThe autoaggregation assay was performed according to Del Re et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Briefly, the selected LAB grown for 48 h at 37\u0026deg;C in MRS broth, were then centrifuged for 10 min at 10000 xg. Subsequently, cells were washed three times with phosphate saline buffer (PBS). Further, obtained cells were suspended in sterile PBS to obtain a 10\u003csup\u003e8\u003c/sup\u003e CFU/mL concentration. A suspension of 5 mL was vortexed for 10 s and incubated at room temperature for 24 h. After this time, the OD at 600 nm was measured. The percentage of autoaggregation was expressed as follows:\u003c/p\u003e \u003cp\u003eAutoaggregation (%) = [(OD\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;OD\u003csub\u003e2\u003c/sub\u003e)/OD\u003csub\u003e1\u003c/sub\u003e] \u0026times; 100\u003c/p\u003e \u003cp\u003eWhere OD\u003csub\u003e1\u003c/sub\u003e and OD\u003csub\u003e2\u003c/sub\u003e are the optical densities at the initial time and after 24 h, respectively. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eResistance to gastrointestinal passage and antimicrobial properties of the strains\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eAcid tolerance\u003c/h2\u003e \u003cp\u003eThe acid tolerance of the isolated strains was evaluated by inoculating active cultures in in MRS broth with pH adjusted to 2.5 with HCl 1N. Cell viable counts were done initially and followed by incubation at 37\u0026deg;C for 1.5 and 3 h. At each time, decimal dilutions were plated in MRS agar to determine the colony-forming units per mL (CFU/mL). The survival percentage was calculated as the relation between the final Log CFU/mL and the initial Log CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBile tolerance\u003c/h2\u003e \u003cp\u003eThe bile tolerance of the selected LAB strains was evaluated by incubating bacterial culture in MRS broth containing 0.3%w/v bile salts (Sigma-Aldrich, USA) at 37\u0026deg;C for 24 h. An initial and final viable count was done in MRS agar. The survival percentage was calculated as the relationship between the final Log CFU/mL with respect to the initial Log CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTolerance to simulated gastrointestinal conditions\u003c/h2\u003e \u003cp\u003eThe selected LAB strains grown in MRS at 37\u0026deg;C for 48 h were harvested and resuspended in a simulated gastric juice (NaCl 125 mM, KCl 7 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 45 mM, pepsin 3 g/L, pH adjusted to 2.5) at OD 600 nm 0.5 (10\u003csup\u003e8\u003c/sup\u003e CFU/mL). The suspensions were incubated at 37\u0026deg;C for 1.5 h and then centrifuged 10 min 10000 x\u003cem\u003eg\u003c/em\u003e. The bacterial pellet was then resuspended in simulated intestinal fluid (NaCl 22 mM, KCl 3.2 mM, NaHCO\u003csub\u003e3\u003c/sub\u003e 7.6 mM, pancreatin 0.1%w/v, bovine bile salts 0.15%w/v, final pH adjusted to 8.0) and incubated at 37\u0026deg;C for 2.5 h. After treatment, samples were diluted in saline solution and plated on MRS agar to determine bacterial viability (Grimoud et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The percentage of survival of the strains was calculated as the relation between Log CFU/mL after and before the sequential gastrointestinal treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial activity\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eDouble layer agar method\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of the selected LAB strains against the foodborne pathogens \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 11229 and \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 10876 was determined using the double-layer agar method, as described by Ripamonti et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Overnight test cultures were spotted (5 \u0026micro;L) on the surface of MRS agar and incubated for 24 h at 37\u0026deg;C, and two different assays were carried out. In the first one, the LAB colonies grown in the surface of MRS agar plates were inactivated with chloroform for 30 min. Then 10 mL of brain heart infusion (BHI, Britania, Argentina) soft agar (0.7%w/v) inoculated with 50 \u0026micro;L of an overnight culture of each pathogen indicator grown in BHI was poured onto MRS agar plates. In the second one, the LAB colonies were not inactivated. The plates with double-layer agar were incubated aerobically at 37\u0026deg;C for another 24 h and the inhibition zones were subsequently measured (cm).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDiffusion agar assay\u003c/h2\u003e \u003cp\u003eEvaluation of the antagonic activity of the LAB culture supernatants was screened by the agar well diffusion assay reported by Tejero-Sari\u0026ntilde;ena et al., (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with some modifications. Briefly, Petri dishes were overlaid with 20 mL of BHI agar previously inoculated with 100 \u0026micro;L of an overnight culture of the foodborne pathogen indicator microorganisms grown in BHI broth (\u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eB. cereus\u003c/em\u003e). Subsequently, cell-free supernatant, sterilized by filtration with 0.22 \u0026micro;m pore size membrane (Gamafil, Argentine), were placed into agar wells (40 \u0026micro;L). Also, cell-free supernatants neutralized with NaOH 3N and 20-fold concentrated by lyophilization were analyzed. The plates were incubated aerobically for 24 h at 37\u0026deg;C and the inhibition zones were subsequently measured (cm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSafety evaluation of selected LAB strains\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eAntibiotic susceptibility\u003c/h2\u003e \u003cp\u003eThe following antibiotics were tested at the recommended minimal concentration (mg/L) for homofermentative/heterofermentative \u003cem\u003eLactobacillus\u003c/em\u003e strains: ampicillin (1/4), gentamicin (16/16), streptomycin (16/64), erythromycin (1/1), clindamycin (1/1), tetracycline (4/8) and chloramphenicol (4/4) (EFSA FEEDAP Panel, 2012). All antibiotics were dissolved in the appropriate diluent for preparing concentrated stock solutions (10X). Then, stock solutions were diluted 1/5 in LSM broth (90% Iso-Sensitest plus 10% MRS). Bacterial cultures growth 48 h at 37\u0026deg;C were diluted 1:50 in LSM broth for inoculation of microdilution plates. Then, 100 \u0026micro;L of diluted inoculum in LSM were added to each well containing 100 \u0026micro;L of an antibiotic solution in LSM (2X). After incubating plates under aerobic conditions at 37\u0026deg;C for 48 h, OD at 600 nm was measured. The susceptibility of LAB strains was categorized as resistant (R) when the growth was higher than 10% concerning the control media without antibiotic, moderately sensitive (MS) when the growth was lower than 10%, or susceptible (S) when no growth was observed.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHemolytic activity\u003c/h2\u003e \u003cp\u003eThe selected LAB cultures were streaked on blood agar (Britania, Argentine) and incubated for 72 h at 37\u0026deg;C under aerobic conditions. Green zones around the colonies suggested α-hemolysis, clear zones around the bacterial colonies indicated the presence of β-hemolysis, whereas no clear zones were recorded as non-hemolytic.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDNAse activity\u003c/h2\u003e \u003cp\u003eThe selected LAB isolates were streaked onto a deoxyribonuclease (DNAse) agar medium (Britania, Argentine) to test for the production of the DNAse enzyme. The plates were incubated at 37\u0026deg;C for 48 h under aerobic conditions and observed for the zone of DNAse activity after the addition of HCl 1N. A clear zone around the colonies was considered as positive (+) DNAse activity, whereas no clear zones were recorded as negative (-) (Shuhadha et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eIsolated bacterial strains\u003c/h2\u003e \u003cp\u003eAfter seeding FYP CaCO\u003csub\u003e3\u003c/sub\u003e agar plates with serial dilutions of the enriched medium, many colonies surrounded by a clear zone due to the local solubilization of CaCO\u003csub\u003e3\u003c/sub\u003e by presumptive lactic acid bacteria from the rhizosphere of the tubers, were obtained. Forty-eight colonies were isolated, 35 cocci and 13 bacilli, Gram positive, catalase and oxidase negative. Considering the acidification power in FYP broth, 3 cocci (named as A, E, J) and 3 rods (named as F, G, H) were selected for their further characterization and assays of their probiotic capacity as a preliminary step for their potential use in the development of vegetal-fermented food. Neither of the selected isolated strains were able to grow at 45\u0026deg;C nor with NaCl 4,5 or 6%w/v. The 3 strains with coccus morphology did not produced gas from glucose, while the 3 rod-shaped strains produced gas from glucose, indicating homofermentative and heterofermentative metabolism, respectively. The colony macroscopic aspect in MRS agar and microscopic morphology subjected to a Gram stain of the selected strains grown at 37\u0026deg;C are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A difference in the bacterial morphology in liquid and solid medium was observed for the 3 selected rod-shaped strains. They presented a typical bacilli shape in the broth, while in solid medium they grew forming shorter bacilli cells. Meanwhile, the coccus-shaped strain presented the same bacterial morphology when they grew in MRS broth or solid media. All the strain grew in MRS broth at 37\u0026deg;C, the pH decreased during bacterial growth, and they reached the stationary phase in 24 h with concentrations around 9 Log CFU/mL (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The acidification in the MRS broth of the cocci was slightly higher than that of the bacilli, reaching final pH values of 3.6\u0026ndash;3.8 and 4.1\u0026ndash;4.3 after 48 h, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The three rod-shaped strains were able to ferment glucose and fructose, while the three coccus-shaped strains, in addition to these carbohydrates, they also fermented sucrose and mannitol. The isolated strains revealed no considerable casein degradation during their aerobic growth in milk agar plates. The isolated strains A, E and J showed autoaggregation capacity after 24 h of 62.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5; 58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 and 71.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9% respectively; whereas the strains F, G and H presented values of 77.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7; 61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4 and 81,5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2%, respectively.\u003c/p\u003e \u003cp\u003eIn this instance the focus was placed in the rod-shaped strains. The amplification products corresponding to the 16S RNA gene gave an approximately 1400 bp amplicon. The analysis of the sequences obtained, revealed similarity of the strains F, G and H with isolates belonging to the Phylum \u0026acute;Firmicutes\u0026acute;, class \u0026acute;\u003cem\u003eBacilli\u0026acute;\u003c/em\u003e, order \u0026acute;\u003cem\u003eLactobacillales\u0026acute;\u003c/em\u003e, family \u0026acute;\u003cem\u003eLactobacillaceae\u003c/em\u003e\u0026acute;, genus \u0026acute;\u003cem\u003eLentilactobacillus\u003c/em\u003e\u0026acute;. Within this genus, both strains F and H, showed high similarity with strains belonging to the species \u003cem\u003eLentilactobacillus kosonis\u003c/em\u003e, with a percentage of similarity 94.2% (percentage of identity of 96.1%) and 96.4% (percentage of identity 97.3%), followed by \u003cem\u003eLentilactobacillus curieae\u003c/em\u003e percentage of similarity 92.3% (percentage of identity of 96.1%) and 91.8% (percentage of identity 95.6%), for the strains F and H, respectively. Meanwhile the percentage of similarity of the strain G with strains belonging to the species \u003cem\u003eLentilactobacillus kosonis\u003c/em\u003e and \u003cem\u003eLentilactobacillus curieae\u003c/em\u003e was 91.2% (percentage of identity 92.1%). Likewise, percentage of similarity higher than 90% were also obtained concerning other species from the genus \u003cem\u003eLentilactobacillus\u003c/em\u003e, such as \u003cem\u003eLentilactobacillus senioris\u003c/em\u003e and \u003cem\u003eLentilactobacillus fungorum\u003c/em\u003e for the strain G, and \u003cem\u003eLentilactobacillus sunkii\u003c/em\u003e, \u003cem\u003eLentilactobacillus otakiensis\u003c/em\u003e, \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e and \u003cem\u003eSecundilactobacillus odoratitofui\u003c/em\u003e for the strain H.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eProbiotic properties\u003c/h2\u003e \u003cp\u003eAll the strains survived in acidic conditions, in presence of bile salt, and after the simulated sequential gastrointestinal treatment (GIT) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After 1.5 h of exposure at pH 2.5, the percentage of survival were higher than 94.8%, except for the strain J, which presented a significantly lower survival. Meanwhile, after 3 h, the rod-shaped strains presented a significantly higher percentage of survival than the coccus-shaped strains. After the bile salt exposure, the rod-shaped strains presented high resistance (\u0026gt;\u0026thinsp;94.2%) without significant differences in their percentage of survival. Among the isolated coccus, the strain A show survival percentage after bile salt treatment of 80.2%, with no significant differences with the strain J, and significantly higher than the strain E. A high resistance was also observed after the sequential simulated GIT challenge for all the selected strains. The rod-shaped strains presented significantly higher survival than the coccus-shaped strains.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of bacterial survival after acidic treatment (pH 2.5, 37\u0026deg;C, 1.5 h or 3 h), bile salt exposure (0.3%w/v, 37\u0026deg;C, 24 h) and simulated sequential gastrointestinal treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcid treatment\u003c/p\u003e \u003cp\u003e1.5 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcid treatment\u003c/p\u003e \u003cp\u003e3 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBile salt treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGIT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoccus-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 \u003csup\u003eB b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1 \u003csup\u003eA a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 \u003csup\u003eAB b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e74.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 \u003csup\u003ea A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u003csup\u003eB b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 \u003csup\u003eA a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4 \u003csup\u003eA a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 \u003csup\u003ea A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 \u003csup\u003eA a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 \u003csup\u003eB b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2 \u003csup\u003eA ab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 \u003csup\u003ea A\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRod-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44 \u003csup\u003eB a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 \u003csup\u003eC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 \u003csup\u003eC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u003csup\u003eb C\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u003csup\u003eB a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 \u003csup\u003eC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9 \u003csup\u003eC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003eb BC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u003csup\u003eB a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 \u003csup\u003eC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e94.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 \u003csup\u003eBC a\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 \u003csup\u003ea B\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eGIT: Gastrointestinal treatment. Values are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of at least three independent replicates. Superscripts with different capital letters indicate significant differences between strains, for each column. Lowercase superscripts indicate significant differences between the cocci-shaped or rod-shaped strains, for each column. ANOVA followed by Tukey test (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBoth viable cells and the metabolites of all the selected strains exerted an inhibitory effect on the pathogenic microorganisms \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e. Moreover, this antimicrobial activity was significantly higher for the rod-shaped strains than that observed for the coccus-shaped strains. The agar plates with inhibition zones produced by the strains F, G, and H against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The strain F exerted an inhibitory effect significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) against both pathogens than the rest of the strains, with diameters of inhibition of 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 cm and 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, respectively. The lowest inhibition was exerted by the strain A against \u003cem\u003eB. cereus\u003c/em\u003e (diameter of inhibition 1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 cm) and strains A and J with \u003cem\u003eE. coli\u003c/em\u003e (diameters of inhibition 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 cm). The inhibition exerted by metabolites of rod-shaped strains against \u003cem\u003eB. cereus\u003c/em\u003e did not show significant differences (diameters of inhibition between 4.1 and 4.4 cm). Finally, the inhibition against \u003cem\u003eE. coli\u003c/em\u003e exerted by the rod-shaped strains produced diameters between 3.9 and 4.3 cm. On the other hand, the concentrated and neutralized culture supernatants of any of the selected strains show inhibitory effect against the pathogens analyzed (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eSafety aspects\u003c/h2\u003e \u003cp\u003eNone of the selected strains showed hemolytic capacity or DNAse activity. These attributes indicated safety aspects for these strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, the isolated strains were sensitive to the antibiotic ampicillin, streptomycin, erythromycin, clindamycin, gentamycin, tetracycline and chloramphenicol, at concentrations below the cut-off limit established by the EFSA (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), for heterofermentative and homofermentative \u003cem\u003eLactobacillus\u003c/em\u003e strains and therefore they could be classified as sensitive (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), however a moderate sensitivity was observed for the three rod-shaped strains against tetracycline.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibiotic sensibility, hemolytic and DNAse activity of the isolated strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSTR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eERT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCLI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGEN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTET\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCLO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHemolytic capacity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eDNAse activity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCoccus-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRod-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eS: sensitive. MS: moderately sensitive. R: Resistant. (-): Negative. (+): Positive.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe rhizosphere is an interesting environmental niche enriched in plant secretions and soil-associated bacteria and fungi. Thus, it can be thought as a potential source for isolating competitive bacterial strains exhibiting probiotic properties in the human gastrointestinal tract (Singhal et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The strains A, E and J resulted Gram positive coccus, catalase and oxidase negative, homofermentative, unable to growth at 45\u0026deg;C nor with NaCl 6%w/v, and gamma-hemolytic, so they could be classified as \u003cem\u003eStreptococcus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe genus formerly called \u003cem\u003eLactobacillus\u003c/em\u003e displays a great level of genetic diversity. The reclassification of the formerly \u003cem\u003eLactobacillus\u003c/em\u003e genus in 23 new genera proposed in 2020 by Zheng et al. (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was considered for classifying the rod-shaped isolated strains. The molecular analysis of the strains F, G and H allowed them to be classified as members of the genus \u003cem\u003eLentilactobacillus\u003c/em\u003e. In recent years, strains with probiotic potential that were identified within the genus \u003cem\u003eLentilactobacillus\u003c/em\u003e have been isolated and from vegetable and fermented food sources (Carasi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ebrahimi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The genus \u003cem\u003eLentilactobacillus\u003c/em\u003e is mainly represented by Gram-positive, rod-shaped, catalase negative, heterofermentative strains, that mostly grow at 15\u0026deg;C and some also grow at 45\u0026deg;C. Most of the strains that constitute this genus were isolated from silage, fermented vegetables, wine and cereal mashes (Zheng et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All these sources present environmental characteristics that can be considered similar to those of the Jerusalem artichoke tubers. Moreover, strains in this genus lead a free-living lifestyle, and generally metabolize a broad spectrum of pentoses, hexoses, and disaccharides. The highest percentage of similarity found in this work was 96.4% for the isolate referred as H with the strain \u003cem\u003eLentilactobacillus kosonis\u003c/em\u003e strain C06.No73T, isolated from a traditional Japanese fermented beverage called k\u0026ocirc;so (Chiou et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), closely related also with the previously described \u003cem\u003eLentilactobacillus curieae\u003c/em\u003e CCTCC M 2011381T from stinky tofu (Lei et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The similarity percentage of the isolated strains F and G were below 96%, being the highest 94.2% with \u003cem\u003eLentilactobacillus kosonis\u003c/em\u003e and 91.2% with \u003cem\u003eLentilactobacillus kosonis\u003c/em\u003e and \u003cem\u003eLentilactobacillus curieae\u003c/em\u003e, for F and G, respectively. Considering that a percentage of similarity higher than 90% was observed concerning several species, such as \u003cem\u003eLentilactobacillus senioris\u003c/em\u003e, \u003cem\u003eL. fungorum\u003c/em\u003e, \u003cem\u003eL. sunkii\u003c/em\u003e, \u003cem\u003eL. otakiensis\u003c/em\u003e, \u003cem\u003eL. buchneri\u003c/em\u003e and \u003cem\u003eSecundilactobacillus odoratitofui\u003c/em\u003e, with which they also share ecological and metabolic properties, however other molecular studies could contribute to specify the identification of the strains at the species level (Zheng et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the base of the 16S rRNA gene sequence Lei et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) showed that the strain \u003cem\u003eL. cuerieae\u003c/em\u003e CCTCC M 2011381T was closely related to \u003cem\u003eL. senioris\u003c/em\u003e JCM 17472T, similar results with the obtained in the present work for rod-shaped strains isolated form Jerusalem artichoke tubers. It is worth to mention that the strain \u003cem\u003eL. curieae\u003c/em\u003e CCTCC M2011381 showed a great potential as starter culture in fermentation of plant foodstuff (Liu et al., 2019). In another hand, \u003cem\u003eLentilactobacillus buchneri subsp. buchneri, Levilactobacillus brevis\u003c/em\u003e, and \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e were isolated and identified from Jordanian traditional pickled and fermented foods (Abudoleh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe autoaggregation capacity can favor the bacterial adhesion to intestinal epithelial cells, allowing bacteria to form a barrier and prevent the adhesion of undesirable microorganisms (Saito et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, a high autoaggregation capacity is desirable for potential probiotic candidates. As suggested by Kos et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) the autoaggregation capacity is related to the type of proteins on the cell surface. The percentages of autoagregation obtained for the strains isolated from Jerusalem artichoke tubers in this work were high (\u0026gt;\u0026thinsp;50%). Among the isolated strains, the highest autoaggregation capacity was observed for the strain H (81,5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2%) followed by the strain F (77.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7%). In general, lower percentage of autoagreggation were reported for LAB strains isolated from different vegetable sources. Fonseca et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that LAB strains isolated from food and fermented vegetable foods, presented percentage of autoaggregation from 16.5 to 52.6%. Various LAB strains isolated from ethnic pickled bamboo shoot products displayed values of autoaggregation between 20 and 55%, in particular the strains \u003cem\u003eLv. brevis\u003c/em\u003e FS2.1, \u003cem\u003eP. pentosaceus\u003c/em\u003e FS36.2, \u003cem\u003eLb. argentoratensis\u003c/em\u003e FS40.1, and \u003cem\u003eP. pentosaceus\u003c/em\u003e FS34.1, showed the highest values (Kanpiengjai et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, percentage of autoaggregation ranging from 2 to 28%, were reported for LAB strains isolated from fermented grains of Chinese baijiu (Fan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The high autoggregation obtained for LAB strains isolated from the Jerusalem artichoke tubers represent a positive aspect for their survival in adverse conditions resulting from a lesser exposure and also favorable for their intestinal adhesion capacity. On another hand, the selected LAB strains isolated in this work were able to ferment only a few carbohydrates, and as expected, the strains do not show proteolytic capacity.\u003c/p\u003e \u003cp\u003eThe survival of strains after the passage through the gastrointestinal tract (GIT), high acidic and bile salt levels (Han et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Urdaneta \u0026amp; Casades\u0026uacute;s, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as well as the adhesion to epithelial cells capacity (Garcia-Gonzalez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Petrova et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), are important preliminary tests to claim about potential probiotic properties of a microorganism (Krawczyk et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, no scientific consensus exists on the pH and bile concentration to which probiotic strains should be tolerant (Zago et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The survival rates after acidic, bile salts, and simulated gastrointestinal treatments for the isolated strains were high. This assay demonstrated the resistance of the strains against adverse environments and their potential ability to reach the colon in high concentrations when consumed as part of food. Similar results were reported by other authors for LAB strains isolated from different natural sources (Delgado et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ramos et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vinderola et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zago et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In particular, Singhal et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that \u003cem\u003eLactiplantibaciullus plantarum\u003c/em\u003e strains isolated from rhizospheric soil remained viable in the presence lysozyme, after simulated gastric juice, and moderate bile salts exposure. Also, the strain \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e subsp. \u003cem\u003emesenteroides\u003c/em\u003e isolated from fermented carrot ang ginger brine exhibited high tolerance to acid, bile, and lysozyme (Cele et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Abudoleh et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported only 1\u0026ndash;2 Log CFU/mL reduction after simulated GIT for LAB strains isolated from plant-based fermented foods. Also, LAB strains isolated from a naturally fermenting coconut palm nectar could resist the simulated GIT without losing viability (Somashekaraiah et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In line, many LAB strains isolated from Neera, resisted to simulated GIT and presented good antimicrobial activity (Somashekaraiah et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe selected LAB strains of the present work showed a remarkable antimicrobial activity against the pathogenic bacteria \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, without producing bacteriocin-like substances, like most of the probiotic candidates isolated from unconventional sources. The inhibition results were attributed to the effect of the organic acids produced during bacterial growth, since no inhibition was exerted by the concentrated and neutralized culture supernatants, suggesting that they did not produce bacteriocin-like substances. In concordance, Fhoula et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that culture supernatants of LAB isolated from rhizosphere exhibited strong inhibitory activity against several pathogenic bacteria, but only a few neutralized supernatants showed antagonistic activity against the tested pathogens. Bacterial strains isolated from traditional fermented Indian food, identified as \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e 86 and \u003cem\u003eWeissella cibaria\u003c/em\u003e 92, showed considerable antimicrobial activity against Gram-positive and Gram-negative pathogens (Patel et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In contrast, Min Hsiu et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) informed that beneficial LAB isolated from fresh fruits and vegetables produced bacteriocin and other antimicrobial substances active against pathogenic bacteria and fungi.\u003c/p\u003e \u003cp\u003eSafety aspects are essential when selecting probiotic candidates to support their potential use in food applications. In the present work, the selected LAB strains were sensitive to antibiotics for human and animal use, complying with European regulated cut-off values. Other authors reported that LAB isolates from Neera were resistant to some of the antibiotics tested; however, it is accepted that it is rarely their risk of infection outside healthcare situations (Sanders et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, Arias and Murray, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and Hanchi et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that plant-origin strains generally display low virulence levels. It is important to remark that before using new isolates in food or feed formulations, their virulence and antimicrobial resistance genes must be verified to prevent the horizontal gene transfer for antibiotic resistance (EFSA FEEDAP Panel, 2018). Probiotic properties, safety aspects, and beneficial health effect must be reinforced by other \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies, if strains are intended to be used for food applications. Finally, this research confirms that the plant rhizosphere might be explored as a valuable ecological niche representing a source of strains with essential and desirable probiotic potential, that promote their use in plant-based foods.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, in this work, lactic acid bacteria were isolated from an unexplored source, such as the Jerusalem artichoke tubers, for the first time, and results showed that they were a reservoir of promising probiotic candidates. The results proved that the selected isolated LAB exhibited a high survival rate in the simulated gastrointestinal treatment, with non-hemolytic activity and antibiotic sensitivity. The isolated strains also showed antimicrobial activity against pathogen microorganisms, mainly attributed to their acidification capacity. The molecular identification of the bacilli strains showed a high similarity with the genus \u003cem\u003eLentilactobacillus\u003c/em\u003e and, within this genus, with the species \u003cem\u003ekosonis\u003c/em\u003e and \u003cem\u003ecurieae\u003c/em\u003e. Hence, these strains revealed potential probiotic \u003cem\u003ein vitro\u003c/em\u003e characteristics that position them to be used in plant-based functional food. The promising results promote further studies on the \u003cem\u003ein vivo\u003c/em\u003e probiotic potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCI, IR and AGA are researcher from the National Council of Scientific and Technological Research (CONICET, Argentina) and GDM is researcher from de Commission of Scientific Investigations (CIC, Argentina). Authors are grateful for the financial support received from the research project PICT2019-0211 of the National Agency for Promotion of Scientific and Technical Research (ANPyCT-FONCyT, Argentina).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the research project PICT2019-0211 of the National Agency for Promotion of Scientific and Technical Research (ANPyCT-FONCyT, Argentina).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by C. Iraporda and I.A. Rubel. The first draft of the manuscript was written by C. Iraporda and I.A. Rubel all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work did not require ethics approval.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbudoleh, S.M., Hamdan, S.O., Mahasneh, A.M., Al-Khani, Z.M., \u0026amp; Talhouni, A.A. (2021). Isolation and characterization of potential probiotic bacteria from jordanian traditional pickled and fermented foods. \u003cem\u003eActa Poloniae Pharmaceutica\u003c/em\u003e, 78(4), 515-520. DOI: 10.32383/appdr/141300\u003c/li\u003e\n\u003cli\u003eArias, C.A. \u0026amp; Murray, B.E. (2012). 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Food-origin lactic acid bacteria may exhibit probiotic properties: Review. \u003cem\u003eBioMed Res. Int\u003c/em\u003e., 5063185. https://doi.org/10.1155/2018/5063185\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Jerusalem artichoke (Helianthus tuberosus L.) tubers, lactic acid bacteria, molecular identification, probiotic potential, safety","lastPublishedDoi":"10.21203/rs.3.rs-3976150/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3976150/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe search for probiotic candidates is an area that accompanies the world trend of development of novel probiotic strains and new products. In recent years, unconventional sources of potential probiotic bacteria have been studied. Furthermore, nowadays there has been a growing interest in non-dairy probiotic products and fermented plant-based foods, which has led to the development of probiotic foods currently being presented as a research priority for the food industry. The aim of this work was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from Jerusalem artichoke (\u003cem\u003eHelianthus tuberosus\u003c/em\u003e L.) tubers. The results proved that the selected isolated LAB strains exhibited a high survival rate in the simulated gastrointestinal treatment, with non-hemolytic nor DNAse activity and antibiotic sensitivity. The isolated strains also showed antimicrobial activity against pathogen microorganisms, due to their acidification capacity. The molecular identification of the bacilli strains showed a high similarity with the genus \u003cem\u003eLentilactobacillus\u003c/em\u003e and, within this genus, with the species \u003cem\u003ekosonis\u003c/em\u003e and \u003cem\u003ecurieae\u003c/em\u003e. Hence, these strains revealed potential probiotic \u003cem\u003ein vitro\u003c/em\u003e characteristics that position them to be used in plant-based functional food.\u003c/p\u003e","manuscriptTitle":"Lactic acid bacteria strains isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers as potential probiotic candidates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-26 10:21:24","doi":"10.21203/rs.3.rs-3976150/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5dce3b0-18dd-46c5-8415-8cd657311f3d","owner":[],"postedDate":"February 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-11T09:36:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-26 10:21:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3976150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3976150","identity":"rs-3976150","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}