Probiotic and technological potential of Enterococcus faecium EM03, isolated from Brazilian semi-hard cheese

Probiotic and technological potential of Enterococcus faecium EM03, isolated from Brazilian semi-hard cheese | 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 Probiotic and technological potential of Enterococcus faecium EM03, isolated from Brazilian semi-hard cheese Emanuela Stefany Oliveira Maciel, Giovanni Eiji do Nascimento Ozaki, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8297099/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Probiotics have attracted increasing interest from consumers due to their potential health benefits, antimicrobial properties, and overall wellness support. In this context, it is essential to identify new bacterial strains that exhibit significant functional characteristics and enhanced stability to food processing. This study aimed to identify and assess the technological and probiotic properties of Enterococcus faecium EM03, which was isolated from semi-hard cheese commercialized in Barreiras, Bahia, Brazil. The isolate was genotypically confirmed as E. faecium through 16S rRNA gene sequencing. In vitro safety assessments revealed sensitivity to most of the antibiotics tested, with resistance observed only to ciprofloxacin and gentamicin. Molecular analyses indicated the absence of genes associated with the production of biogenic amines and several virulence factors. Conversely, the presence of genes linked to bacteriocin production, specifically ent B, ent P, L50AB, Ian M, and Ian C, was confirmed. These findings were further supported by results from the spot-on-the-lawn assay, which demonstrated that E. faecium EM03 inhibited the growth of Listeria monocytogenes and E. faecalis through the production of proteinaceous compounds. Regarding its probiotic potential, the isolate exhibited high survival rates under simulated gastrointestinal conditions, including tolerance to pH 3.5, to pepsin treatment, and to bile salts. Additionally, microencapsulation by spray-drying allowed the survival to in vitro gastrointestinal tests and preserved probiotic viability for up to 60 days, with stable physical properties of the microparticles. Overall, the results indicate E. faecium EM03 has technological potential for food and health-related applications. Bacteriocins Functional Products Lactic Acid Bacteria Microencapsulation Spray-drying Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Probiotics are defined by the the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) [ 1 ] as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”. Probiotic bacteria comprise mainly lactic acid bacteria (LAB), which are non-sporulating, Gram-positive, catalase-negative cocci, coccobacilli or rods. LAB can produce lactic acid via carbohydrate metabolism besides other antimicrobial compounds synthesized by diverse pathways. In addition, LAB may offer a protective barrier for the intestinal mucosa and promote several health benefits, including modulation of the immune system [ 2 ]. The beneficial effects of LAB have been widely investigated but several mechanisms of action remain inconclusive [ 3 ]. Lactobacillus , Bifidobacterium , and Enterococcus are the main genera of LAB [ 4 – 6 ], which are naturally found in foods and as commensals in the gastrointestinal tract of humans and animals. The genus Enterococcus includes a total of 19 cataloged species, according to chemotaxonomic and phylogenetic studies, with Enterococcus faecium being one of the most extensively studied species [ 7 ]. E. faecium is able to produce bacteriocins (ribosomally synthesized antimicrobial peptides) and bile salt hydrolase (BSH), besides being able to survive under gastrointestinal conditions such as high acidity, presence of bile salts, and digestive enzymes. These traits aid enterococci to reach and colonize the intestine, where they may exert beneficial effects. Enterococcus sp. also attracts the attention of the food industry as a biopreservative culture [ 8 ]. Despite these promising characteristics, E. faecium is not widely used in food and feed in part due to its association with nosocomial infections and multidrug resistance[ 9 ]. Moreover, the gene transfer capability of Enterococcus species is of special concern. The significance of Enterococcus sp. in foods may be related to its role as an indicator of inadequate sanitary conditions. Nevertheless, previous studies [ 7 , 10 , 11 ] affirmed that enterococci that lack virulence factors, can be considered safe for use as probiotics. Therefore, the use of E. faecium for human consumption requires the evaluation of pathogenicity aspects of each strain, according to FAO and WHO [ 1 ]. From this perspective, the present study aimed to identify and to evaluate, through in vitro assays, the probiotic properties of Enterococcus faecium EM03, isolated from a sample of semi-hard cheese commercialized in Barreiras, Bahia, Brazil. The probiotic candidate strain was also microencapsulated by spray-drying and evaluated with regard to its technological potential. 2. Methodology 2.1 Bacterial strains E. faecium EM03 was isolated from semi-hard cheese. The cheese samples were homogenized in a 0.9% (w/v) NaCl solution, tenfold diluted, and plated onto MRS agar. Following incubation at 37°C under anaerobic conditions, typical LAB colonies were selected and subsequently inoculated into MRS broth. One of the isolates was identified as a Gram-positive, catalase-negative, cocci-shaped lactic acid bacterium. It was chosen for further characterization due to its antilisterial activity. Other strains used in this study are listed on Table 1 . All the strains were maintained at -40°C in BHI or MRS broth (Kasvi, Brazil) containing 20% (v/v) glycerol (Synth, Brazil). Table 1 Strains used during this study. Organism Source Culture Media Incubation temperature Escherichia coli NCTC 11954 National Collection of Type Cultures a BHI b 37°C Enterococcus faecalis NCTC 12967 National Collection of Type Cultures BHI 37°C Enterococcus faecium EM03 This study BHI 37°C Klebsiella pneumoniae NTC 13368 National Collection of Type Cultures BHI 37°C Listeria monocytogenes NCTC 13627 National Collection of Type Cultures BHI 37°C Pediococcus acidilactici LK08 Our collection MRS c 37°C Pseudomonas aeruginosa NCTC 12903 National Collection of Type Cultures BHI 37°C Salmonella Enteritidis NCTC 6679 National Collection of Type Culture BHI 37°C Staphylococcus aureus NCTC 12493 National Collection of Type Cultures BHI 37°C a National Collection of Type Cultures , United Kingdom. b BHI broth ( Brain Heart Infusion ). c MRS broth (De Man, Rogosa, and Sharpe). 2.2 Genotypic identification Genotypic identification was performed by sequencing the 16S rRNA gene and for this purpose, genomic DNA was extracted using the kit Wizard DNA Genomic Purification (Promega, Madison, WI, USA). Next, the samples were amplified and purified using the kit Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA). The DNA extracted was used as a template for identification based on the polymerase chain reaction (PCR) carried out in a third-party laboratory, using the primer forward 27F [ 12 , 13 ] from Thermo Fisher Scientific, São Paulo, Brazil. Purified PCR products were sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems, USA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were analyzed using Chromas Lite 2.1 (Technelysium, South Brisbane, Australia) and compared with sequences available in GenBank using the BLASTN search program from the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov/BLAST ). 2.3 Genotypic characterization Total genomic DNA of E. faecium EM03 was extracted from a 24 h BHI culture heated at 55°C for 2 h with agitation. Cells were collected by centrifuging 1.5 mL of growth medium at 10,000 x g for 1 min, then resuspended in 200 µL of ALR-PK buffer with 4 µL of lysozyme. After 90 min of incubation at 56°C, DNA was extracted using the SAMPLE FLEX phT kit (Phoneutria NA2110, Sigma-Aldrich, Missouri, USA) as per the manufacturer’s instructions. Extracted DNA quality was checked by spectrophotometry with a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Massachusetts). Samples were stored at − 20°C for later use. Gene prevalence was determined by PCR for each gene group described in Table 2 . The amplification reaction included 1 µL of genomic DNA, 2.0 µL of 10X buffer, 0.2 mM of each dNTP (Cellco, Brazil), 1 U Taq DNA polymerase (Cellco), and 1 µL of a primer pair mixture in a 20 µL final volume. Amplification began with 5 min at 94°C, followed by 30 cycles of 60 s at 95°C, 60 s at the group-specified annealing temperature (Table 2 ), and 60 s at 72°C. The reaction concluded with a final extension of 10 min at 72°C. Amplicons were analyzed by electrophoresis on a 2% agarose gel containing 1 µL of ethidium bromide. Images were captured using an Lpix Image photodocumentation system (Loccus Biotecnologia, Brazil). Table 2 Oligonucleotide sequences of the primers used in this study a . Target genes Oligonucleotide sequences (5’-3’) Annealing temperature (°C) Amplicon (pb) Reference Virulence genes hyl Fw: ACA GAA GAC CTG CAG GAA ATG Rv: GAC TGA CGT CCA AGT TTC CAA 53 276 [ 14 , 15 ] ace Fw: GAA TTG AGC AAA AGT TCA ATC G Rv: GTC TGT CTT TTC ACT TGT TTC 56 1008 [ 16 , 17 ] asa 1 Fw: GCA CGC TAT TAC GAA CTA TGA Rv: TAA GAA AGA ACA TCA CCA CGA 56 375 [ 14 , 16 ] cyl A Fw: ACT CGG GGA TTG ATA GGC Rv: GCT GCT AAA GCT GCG CTT 56 688 [ 14 , 16 ] efa A Fw: GAC AGA CCC TCA CGA ATA Rv: AGT TCA TCA TGC TGT AGT A 54 n.d. [ 16 , 18 ] esp Fw: AGA TTT CAT CTT TGA TTC TTG G Rv: AAT TGA TTC TTT AGC ATC TGG 56 510 [ 14 , 16 ] gel E Fw: TAT GAC AAT GCT TTT TGG GAT Rv: AGA TGC ACC CGA AAT AAT ATA 56 213 [ 14 , 16 ] Biogenic amines genes hdc1 Fw: AGA TGG TAT TGT TTC TTA TG Rv: AGA CCA TAC ACC ATA ACC TT 46 367 [ 15 , 19 ] tdc Fw: GAY ATN ATN GGN ATN GGN YTN GAY CAR G Rv: CCR TAR TCN GGN ATA GCR AAR TCN GTR TG 55 924 [ 15 , 19 ] odc Fw: GTN TTY AAY GCN GAY AAR CAN TAY TTY GT Rv: ATN GAR TTN AGT TCR CAY TTY TCN GG 54 1446 [ 15 , 19 ] Bacteriocin genes pedA Fw: CAA GAT CGT TAA CCA GTT T Rv: CCG TTG TTC CCA TAG TCT AA 50 1044 [ 20 ] lanM Fw: ATG CWA GWY WTG CWC ATG G Rv: CCT AAT GAA CCR TRR YAY CA 40 200–300 [ 15 , 21 ] lanB Fw: TAT GAT CGA GAA RYA KAW AGA TAT GG Rv: TTA TTA IRC AIA TGI AYD AWA CT 40 400–500 [ 15 , 22 ] lanC Fw: TAA TTT AGG ATW ISY IMA YGG Rv: ACC WGK III ICC RTR RCA CCA 40 200–300 [ 15 , 22 ] entA Fw: CAT CAT CCA TAA CTA TAT TTG Rv: AAA TAT TAT GGA AAT GGA GTG TAT 56 126 [ 15 , 23 ] entB Fw: GAA AAT GAT CAC AGA ATG CCT A Rv: GTT GCA TTT AGA GTA TAC ATT TG 58 162 [ 15 , 23 ] entP Fw: TAT GGT AAT GGT GTT TAT TGT AAT Rv: ATG TCC CAT ACC TGC CAA AC 58 120 [ 15 , 23 ] L50AB Fw: STG GGA GCA ATC GCA AAA TTA G Rv: ATT GCC CAT CCT TCT CCA AT 56 98 [ 15 , 23 ] AS48 Fw: GAG GAG TIT CAT GAT TTA AAG A Rv: CAT ATT GTT AAA TTA CCA AGC AA 56 340 [ 15 , 23 ] Species-specif genes Efaecium TAGAGACATTGAATATGCC TCGAATGTGCTACAATC 55 550 [ 16 , 24 ] a Y = C or T; R = A or G; N = A, C, G or T. 2.4 Antibiotic susceptibility test The antibiotic susceptibility of E. faecium EM03 was assessed using the disc diffusion method on MRS agar plates that were inoculated with of approximately 10 5 CFU/mL from an overnight culture of E. faecium EM03 [ 16 , 25 , 26 ]. Antibiotic discs (Laborclin, Pinhais, Brazil) were placed on the surface of the inoculated MRS agar, and the plates were incubated at 37°C for 24 h. For this analysis, it was used antibiotic discs containing amoxicillin (10 µg), ampicillin (10 µg), cefalotin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), gentamicin (10 µg), tetracycline (30 µg), and vancomycin (30 µg). According to the resistance criteria established by Charteris et al. [ 26 ], the strain was classified as resistant if the inhibition zone was less than 18 mm for amoxicillin, 12 mm for ampicillin, 14 mm for cefalotin, 13 mm for chloramphenicol, 13 mm for ciprofloxacin, 8 mm for clindamycin, 13 mm for erythromycin, 12 mm for gentamicin, 14 mm for tetracycline, and 14 mm for vancomycin. 2.5 Evaluation of the probiotic potential 2.5.1 Bacteriocin production and antimicrobial spectrum The antilisterial activity of E. faecium EM03 was assessed using the spot-on-the-lawn method, as described by Lewus et al [ 27 ]. A volume of 2 µL from a 24 h culture grown in MRS Broth (Kasvi, Brazil) was deposited over TSA-ye (supplemented with 0.6% w/v yeast extract) agar plates (Kasvi, Brazil). After incubating the plates at 37 ºC for 48 h under anaerobic conditions (Probac, Brazil), they were overlaid with 7 mL of molten soft BHI agar (0.8% w/v agar) inoculated with approximately 10 6 CFU/mL of bacterial strains listed in Table 1 . Plates were further incubated for 24 h at 37 ºC, and the presence of inhibition halos indicated the production of antimicrobial compounds. To verify whether the antimicrobial compound produced by E. faecium EM03 was a bacteriocin, its susceptibility to proteolytic enzyme degradation was evaluated [ 27 ]. A volume of 2 µL from a 24 h culture grown in MRS Broth (Kasvi, Brazil) was deposited over TSA-ye (supplemented with 0.6% w/v yeast extract) agar plates (Kasvi, Brazil). After incubating the plates at 37 ºC for 48 h under anaerobic conditions (Probac, Brazil), 10 µL of a 20 mg/mL sterile solution of α-Chymotrypsin from bovine pancreas (Sigma-Aldrich, USA) was applied in 2 mm diameter wells on the agar, positioned near the inhibition halo. Water was used as a negative control. The plates were then incubated for 2 h at 25 ºC and, afterward, they were overlaid with 7 mL of molten soft BHI agar (0.8% w/v agar) inoculated with Listeria monocytogenes NCTC 136270 (approximately 10 6 CFU/mL). After 24 h of incubation at 37 ºC, the absence of an inhibition halo around the well containing proteolytic enzyme indicated the proteinaceous nature of the antimicrobial compound ( i.e. bacteriocin). 2.5.2 Resistance to low pH and bile salts The survival of E. faecium EM03 in harsh conditions was evaluated as described by Tulini et al. [ 28 ] with modifications, as this tolerance represents a critical property for probiotic potential. For this assay, 4 mL of a 24 h BHI broth culture was centrifuged at 2,800 xg for 10 minutes. After removing the supernatant, the pellets were washed with 0.9% w/v NaCl solution and resuspended in 4 mL of the following test media: (i) BHI broth adjusted to pH 2.0, 2.5, or 3.5 with acid; (ii) BHI broth containing 0.3% w/v bile salts (Sigma-Aldrich, USA); (iii) 0.9% w/v NaCl solution containing 3 g/L pepsin and adjusted to pH 2.0; (iv) unmodified BHI broth as a control. The test tubes were incubated at 37°C, with samples collected at 0, 90, and 180 minutes. For bacterial enumeration, 100 µL from each sample was serially diluted in 900 µL of 0.9% w/v NaCl solution, and 10 µL from each dilution was plated on BHI agar and incubated at 35°C for 48 h [ 29 ]. The test was performed in triplicate, and survival rates were determined based on colony counts (log CFU/mL). 2.6 Production and characterization of microparticles loaded with E. faecium EM03 Initially, 20 g of Arabic gum and 5 g of inulin were dissolved in 100 mL of distilled water and homogenized using an Ultraturrax (IKA, Staufen, Germany) at 10,000 rpm for 5 minutes. Cell biomass was then harvested from 100 mL of a 24 h BHI broth culture, washed twice with 0.9% w/v NaCl solution, and incorporated into the gum Arabic and inulin solution. The resulting mixture was homogenized at 10,000 rpm for 1 minute using Ultraturrax. Subsequently, the mixture was spray dried using a mini spray dryer (Haurok, Florida, USA) set to an inlet temperature of 140°C, an outlet temperature of 85°C, a feed rate of 10 mL/min (controlled by peristaltic pump), and an airflow rate of 10 L/min, using a 0.7 mm nozzle. The dried product was collected and subjected to characterization analyses. The final yield was assessed by comparing the mass of microparticles collected at the end of the process to the mass of solids incorporated into the formulation. In contrast, the encapsulation efficiency was determined by comparing the total log CFU obtained at the end of the process to the total log CFU present in the formulation. 2.6.1 Moisture content and water activity The moisture content of the powders was determined using a moisture analyzer (BEL Engineering, Piracicaba, Brazil) equipped with infrared radiation. Water activity was measured using a Tecnal Lab Master instrument (Piracicaba, Brazil). 2.6.2 Morphology The morphology of microparticles and carrier was evaluated by scanning electronic microscopy (Tescan Vega 4 LMU, Brno, Czech Republic). The microparticles were placed over pieces of double-faced carbon tape, fixed on aluminum stubs and covered with gold for 2 min. Thereafter, images were acquired using 2 keV and current of 50 pA. 2.6.3 Fourier transform infrared spectroscopy (FT-IR) The powder was evaluated by Fourier transform infrared (FT-IR) spectroscopy in the 4,000 to 400 cm − 1 regions, using a Shimadzu IRAffinity-1S FTIR spectrometer (Kyoto, Japan). 2.6.4 Particle size Particle size distribution of spray-dried powder was determined by laser diffraction using a Mastersizer 2000 (Malvern Instruments, UK) device. Samples were dispersed in ethanol under gentle stirring and diluted until reaching an obscuration of ca. 10%, using the Fraunhofer optical model. All measurements were performed in triplicate. 2.6.5 Resistance to simulated gastrointestinal conditions The resistance of microencapsulated and free E. faecium EM03 cells was assessed using the static INFOGEST in vitro digestion protocol [ 30 ], with modifications. Simulated gastric fluid (SGF) was prepared by dissolving 3 g/L pepsin from porcine gastric mucosa (P7000, Sigma-Aldrich, USA) and 5 g/L sodium chloride in sterile distilled water, and the pH was adjusted to 3.0. Simulated intestinal fluid (SIF) was prepared by dissolving 6.4 g/L sodium bicarbonate, 1.28 g/L sodium chloride, 0.239 g/L potassium chloride, 3 g/L bovine bile salts (B3883, Sigma-Aldrich, USA), and 1 g/L pancreatin from porcine pancreas (P7545, Sigma-Aldrich, USA) in sterile distilled water, with the pH adjusted to 7.0. For microparticle evaluation, 0.1 g of particles was used. As a control, approximately 10⁹ CFU of double-washed E. faecium EM03 cells were included. During the gastric phase, both free cells and microparticles were resuspended in 10 mL SGF and incubated at 37°C. Aliquots of 250 µL were collected at 0, 45, and 90 minutes. Following the gastric phase, samples were centrifuged, the supernatant was removed, and the pellet was resuspended in 10 mL SIF and homogenized. The mixture was incubated at 37°C for the intestinal phase, and aliquots of 250 µL were collected at 0, 60, and 120 minutes. For bacterial enumeration, 100 µL of each sample was serially diluted in 900 µL of 0.9% (w/v) NaCl solution. Subsequently, 10 µL of each dilution was plated on BHI agar and incubated at 35°C for 48 hours [ 29 ]. All experiments were performed in triplicate. Cell survival was reported as log CFU based on colony counts. 2.6.6 Effect of storage temperature Microparticles were stored at 4°C and 25°C for up to 60 days. Samples were collected at 0, 15, 30, 45, and 60 days for bacterial enumeration, as previously described. Water activity, SEM and FT-IR analyses were conducted after 60 days of storage. All experiments were conducted in triplicate, and bacterial survival was expressed as log CFU/g. 2.7 Statistical analyses Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, USA). Differences between groups were evaluated using Student’s t-test for pairwise comparisons and one-way ANOVA to analyze overall group variations, with a significance level set at 5%. Tukey’s post-hoc test was applied following ANOVA to determine specific differences between groups. 3. Results and discussion 3.1 Genotypic identification and characterization The strain analyzed in this study was identified as a Gram-positive, coccoid-shaped, catalase-negative microorganism. Genotypic identification was performed through sequencing of the 16S rRNA gene, leading to its classification as Enterococcus faecium EM03, and confirmed by the detection of the species-specific gene described in Table 1 . In this study, no virulence genes or genes associated with biogenic amine production were detected in E. faecium EM03, with the exception of the hyl gene. This gene encodes hyaluronidase, an enzyme responsible for degrading mucopolysaccharides in connective tissues, which can facilitate the spread of microorganisms or toxins within the host [ 31 , 32 ]. Pathogenicity in Enterococcus strains is usually linked to the presence of multiple virulence genes, along with specific phenotypic traits. The occurrence of these genes can vary depending on the strain being evaluated. Fuka et al. [ 33 ] reported a higher prevalence of virulence genes in E. faecalis strains compared to E. faecium , with genes such as cyl A, efa A, and esp being more common in E. faecalis . In contrast, Tsanasidou et al. [ 34 ] found no presence of the genes ace , esp A, hyl , or gel E in any of the nine E. faecium isolates obtained from food sources. Meanwhile, Kiruthiga et al. [ 35 ] identified the hyl gene exclusively in E. faecium isolates. This variability in genotypic virulence profiles may be attributed to the ability of bacterial strains to undergo horizontal gene transfer. Based on the results of this study and considering the relatively common occurrence of the hyl gene in non-pathogenic Enterococcus strains, it is possible to infer that E. faecium EM03 does not present any pathogenic traits. Therefore, given its safe phenotypic profile and the genotypic characteristics observed in other cheese-derived strains, E. faecium EM03 can be regarded as safe for biotechnological applications. Investigations into the genes responsible for encoding bacteriocins in E. faecium EM03 revealed the presence of the genes ent B, ent P, L50AB, lan M and lan C. The enterocins produced by Enterococcus fall under class II bacteriocins and are noted for their strong antimicrobial activities against foodborne bacteria [ 36 ]. Similar findings were reported by Favaro et al. [ 37 ], who detected a combination of enterocin genes in all analyzed E. faecium strains, including ent A, ent E, ent P, and L50. Additionally, Valledor et al. [ 38 ] explored enterocin genes in E. faecium ST20Kc and ST41Kc, identifying the presence of ent A, ent B, and ent P, which exhibited significant inhibitory effects against L. monocytogenes and E. faecalis . Other studies have similarly documented the co-occurrence of enterocin-encoding genes in E. faecium strains [ 39 – 41 ]. The lan M and lan C genes are implicated in the biosynthesis of lantibiotics, which are peptides that undergo post-translational modifications to attain biological activity, primarily targeting the bacterial cell wall [ 42 ]. Research on these genes is relatively rare among Enterococcus species, despite the prevalence of lantibiotic production in lactic acid bacteria from the genera Streptococcus , Staphylococcus , and Enterococcus [ 43 ]. Although multiple enterocin genes have been confirmed, there is currently no evidence to suggest that all these genes are expressed simultaneously in the examined strain. The detection of several genes may account for the high antimicrobial activity observed against L. monocytogenes and E. faecalis , however, further experiments are necessary to evaluate their actual expression levels. 3.2 Antibiotic Susceptibility E. faecium EM03 exhibited varying susceptibility profiles to the antibiotics tested: it was sensitive to ampicillin (10 µg), tetracycline (30 µg), amoxicillin (10 µg), vancomycin (30 µg), chloramphenicol (30 µg), and cephalothin (30 µg). The strain showed moderate sensitivity to erythromycin (15 µg) and clindamycin (2 µg), while it was resistant to gentamicin (10 µg) and ciprofloxacin (5 µg). Evaluating antimicrobial susceptibility is one of the minimum criteria for considering a strain as a potential probiotic, as in vitro safety assessments are essential [ 44 ]. Antibiotic sensitivity is favorable because it ensures the effective elimination of the microorganism from the host when necessary. Additionally, probiotic candidates should ideally not exhibit significant antibiotic resistance to reduce the risk of transferring resistance genes to the intestinal microbiota. The results indicated high sensitivity of E. faecium EM03 to critically important antibiotics, such as vancomycin and ampicillin, suggesting a low health risk compared to clinical isolates from hospitalized patients. Similar findings were reported by Çetin et al. [ 45 ] for E. faecium strains isolated from raw cow’s milk, which also showed sensitivity to ampicillin, vancomycin, clindamycin, tetracycline, and chloramphenicol, resistance to gentamicin, and intermediate susceptibility to erythromycin. Although antibiotic resistance genes were not investigated in this study, the gentamicin resistance observed in E. faecium EM03 may represent an intrinsic characteristic of the species, as seen in other studies [ 40 , 46 – 48 ] Resistance to glycopeptides, particularly vancomycin and ampicillin, is a significant concern when considering the use of Enterococcus spp. in food applications. This resistance is typically attributed to the presence of peptidoglycan precursors that enhance cell wall rigidity, thereby hindering the effectiveness of these antibiotics [ 48 ]. However, E. faecium EM03 demonstrated a high sensitivity to these glycopeptide antibiotics, indicating the absence of genes associated with peptidoglycan modification, and thereby supporting the efficacy of a broad array of antimicrobials. Corroborating this susceptibility phenotype, Cariolato et al. [ 49 ] reported that E. faecium strains isolated from food sources exhibited greater susceptibility to glycopeptides compared to clinical isolates. Therefore, the differences in resistance patterns observed among Enterococcus spp. strains may be linked to their source of isolation. Clinical strains frequently display multidrug resistance due to the acquisition of resistance genes through conjugation, which is often facilitated by interactions with pathogenic bacteria colonizing the intestinal tracts of hospitalized patients. Resistance to ciprofloxacin observed in some bacterial strains may result from mutations in genes that encode target enzymes affected by the antibiotic, thus reducing its affinity. This association has been documented in Enterococcus species [ 31 ]. Nevertheless, resistance to fluoroquinolones is typically anticipated in E. faecium , primarily due to the production of the chromosomal enzyme aac(6’)-I, which diminishes the effectiveness of aminoglycosides [ 50 ]. In conclusion, E. faecium EM03 demonstrates an antimicrobial susceptibility pattern typical of non-pathogenic strains within the species, displaying no resistance to important antibiotics used in clinical settings and indicating its safety for use as a probiotic. 3.3 Evaluation of the probiotic potential 3.3.1 Bacteriocin production and antimicrobial spectrum The assay performed with E. faecium EM03 exhibited a significant inhibitory effect against Listeria monocytogenes NCTC 136270, producing an average inhibition zone of 22.33 ± 1.15 mm in diameter. Additionally, it demonstrated activity against Enterococcus faecali s NCTC 12967, resulting in a halo measuring 14.33 ± 0.58 mm. However, no inhibitory effect was observed against the other tested indicators. Moreover, it was noted that the antimicrobial compound is of a proteinaceous nature (specifically, a bacteriocin), as indicated by the degradation of the inhibition halo upon treatment with protease (Fig. 1 ). Evaluating the antagonistic activity against pathogens is essential in assessing the functional properties of probiotic candidates. The synthesis of bacteriocins plays a crucial role, as it helps curb the proliferation of pathogenic bacteria and supports the stability of the intestinal microbiota. Previous studies, such as those by Charyyev et al.[ 51 ] and Nami et al. [ 52 ], have reported strong inhibition of L. monocytogenes by various E. faecium strains, with inhibition zones of approximately 23 mm, which aligns closely with the results of the present study. Additionally, inhibitory activity against E. faecalis was noted by Furlaneto-Maia et al. [ 53 ], who reported halos of 10 mm or less. Although these values are lower than those observed in this study, they further emphasize the protective potential of E. faecium . The results affirm the effectiveness of the bacteriocins (enterocins) produced by E. faecium EM03 in inhibiting foodborne pathogens. The notable antilisterial activity observed is characteristic of class IIa enterocins, which are also recognized for their robust resistance to high temperatures [ 8 ]. The mechanisms by which these bacteriocins operate involve altering the permeability of pathogenic bacterial cell membranes, ultimately leading to cell lysis[ 54 ]. In this context, the effects of enterocins have been extensively researched, and their efficacy is viewed as a promising alternative for safeguarding foods against bacterial contamination. Zhou et al. [ 55 ] assessed the enterocins produced by E. faecium B1 in food matrices and reported significant inhibitory activity against L. monocytogenes , comparable to that of the bacteriocins synthesized by E. faecium EM03. Moreover, antilisterial activity has also been demonstrated by Schittler et al. [ 56 ] and Popović et al. [ 57 ], who reported positive outcomes from the use of E. faecium in the production of fermented foods and in the preservation of milk. This efficacy is particularly attributed to its biocontrol capabilities against pathogens and its ability to reduce the viable count of L. monocytogenes . Although the purification and characterization of the bacteriocins produced by E. faecium EM03 were not conducted, the synthesis of these substances is corroborated by the detection of enterocin-encoding genes ( ent A, ent P, L50A, and L50B). Further evaluations conducted by Mancini et al. [ 40 ] and Valledor et al. [ 38 ] on E. faecium species also identified these genes and demonstrated significant antilisterial activity, corroborating the findings of this study. These results underscore the functional potential of E. faecium EM03 as a viable alternative as a protective agent against food related bacteria. This antimicrobial property is advantageous for the selection of probiotics within the Enterococcus genus, enabling specific strains to be utilized for pathogen control in the gastrointestinal tract. 3.3.2 Resistance to low pH and bile salts In the present study, E. faecium EM03 exhibited a significant decline in viability at pH 2.0 during the first 90 minutes, maintaining partial stability up to 180 minutes, which suggests greater sensitivity to extremely acidic conditions. Resistance at pH 2.5 was notably higher than at pH 2.0, with a stable count of 7.3 log CFU/mL during the initial 90 minutes and only slight variation thereafter, indicating mild sensitivity to the gastric environment during prolonged exposure. The highest survival rate was recorded at pH 3.5, with counts ranging from 7.4 to 8 log CFU/mL throughout the 180 minutes, showing some fluctuation up to 90 minutes before stabilizing. The ability to endure acidic conditions and traverse the gastrointestinal tract is one of the primary challenges for probiotic strains. In this context, the gastric environment serves as the first biological barrier, with pH levels ranging from 2.5 to 3.5, thereby limiting bacterial survival [ 58 ]. In the presence of pepsin, the strain demonstrated low resistance during the 90-minute exposure, with counts ranging from 7.3 to 8.5 log CFU/mL. Notably, there was a more significant decline in counts at the end of this period compared to the control group. Conversely, when subjected to 0.3% bile, the count remained stable at approximately 8.5 log CFU/mL over the 180 minutes, showing no significant reductions. This indicates a high viability and adaptation to intestinal conditions (Fig. 2 ). These findings are consistent with studies by Gupta et al. [ 59 ], Ankaiah et al. [ 60 ] and Xiao et al. [ 61 ], which also noted low resistance of E. faecium WFD-128 to extremely acidic environments, indicating that conditions above pH 2.5 are more conducive to survival. In contrast, Bhardwaj et al. [ 62 ] found that some Enterococcus strains exhibited resistance at pH 2.0, though the highest survival rates were observed under milder conditions, particularly between pH 3.0 and 3.5. While certain studies characterize E. faecium as resistant to highly acidic environments [ 63 ], the majority indicate significant growth only at pH values exceeding 2.5. This suggests that E. faecium strains exhibit a high level of acid tolerance, a trait often linked to their ability to maintain pH homeostasis through proton extrusion, thus preserving cell membrane integrity [ 64 ]. Nevertheless, proliferation diminishes as acidity increases, which helps elucidate the growth performance of specific Enterococcus strains in fermented foods. Consequently, E. faecium EM03 shows a considerable ability to survive gastrointestinal passage. Strategies such as microencapsulation or co-administration with foods that can modulate gastric pH may further enhance its viability and probiotic efficacy. The resistance of E. faecium EM03 to pepsin and 0.3% bile enables its effective passage and colonization within the intestinal environment in viable amounts, which is crucial for the expression of probiotic functions. Typically, bile impacts bacterial survival by interacting with surface proteins; however, species from the Enterococcus genus have developed mechanisms to mitigate this effect [ 65 ]. Similar findings were reported by Tilwani et al. [ 66 ], who observed that E. faecium MC-5 maintained its viability, particularly in the presence of 0.3% bile. Likewise, Bhardwaj et al. [ 62 ] noted that E. faecium KH24 did not exhibit significant variations in viability when exposed to various bile concentrations (1%, 2%, and 3%). The bile salt tolerance observed in this study, along with previous research findings, underscores the potential application of E. faecium EM03 as a probiotic. 3.4 Production and characterization of microparticles loaded with E. faecium EM03 Microparticles containing E. faecium EM03 were produced through a spray-drying process, using Arabic gum and inulin as wall materials. The final yield of the microparticles collected from the cyclone compartment of the spray dryer was 39.5%, with an encapsulation efficiency of 94.0% and a bacterial population of 8.53 ± 0.05 log CFU/g. The resulting powder had a moisture content of 5.5 ± 0.4 g/100g and 0.320 ± 0.02 of water activity. Moisture content indicates the total amount of water present in microparticles, while water activity refers to the portion of water that is available for microbial growth and chemical reactions. Both parameters are essential for ensuring the stability of probiotics during storage. Ideally, moisture levels should range between 4% and 7% to maintain stability and prolong the product's shelf life [ 67 ]. According to Barbosa et al. [ 68 ], lower moisture levels can reduce particle agglomeration and promote better dispersion in food matrices. It's also important to highlight that inulin is well-known for its ability to retain water, often forming gel-like structures when hydrated [ 69 , 70 ]. However, its inclusion in the present formulation did not significantly increase moisture content to a level that would adversely affect the rheology of the particles. The optimal water activity (A w ) for probiotics is typically recommended to be below 0.2, as this threshold promotes bacterial survival and mitigates the risk of product contamination during storage [ 71 ]. In this study, the measured A w exceeded the ideal value, but is equivalent to the values reported by Ramírez-Damián et al. [ 72 ], Reyes et al. [ 73 ], and Naseem et al. [ 74 ] who developed encapsulated microparticles using Arabic gum alone or in combination with inulin. Their results revealed water activity similar to those found in this research, ranging from 0.31 to 0.33, which also surpassed the recommended range, probably due to the presence of inulin. The microparticles displayed a primarily spherical morphology, typical of spray-dried systems, as presented in Fig. 3. Their surfaces appear irregular and wrinkled, with several particles exhibiting concavities or collapsed areas, also a common characteristic due to rapid moisture loss during atomization. Additionally, the sample contains numerous smaller satellite particles adhered to the surfaces of larger ones, resulting in an agglomerated structure. Overall, the microstructure indicates effective particle formation, characterized by heterogeneous sizes and typical shrinkage-related depressions observed in polysaccharide-based microcapsules containing inulin and gum Arabic. In 2021, Karrar et al. [ 75 ] encapsulated gurum seed oil by spray-drying using formulations with Arabic gum and whey protein isolate, and obtained very similar results when observing the microparticles by SEM. As shown in Fig. 4 , the Fourier transform infrared spectroscopy (FT-IR) analyses revealed distinct absorption peaks, notably around 3,300 cm⁻¹, corresponding to O–H stretching vibrations. A peak near 1,600 cm⁻¹ is attributed to C = O stretching, while another peak around 1,000 cm⁻¹ suggests C–O stretching or C–O–C vibrations, which are typically associated with glycosidic bonds. The similarities observed between the spectra of the wall material and the microparticles indicate that there was no chemical interaction between the encapsulated material and the wall material. Similar results were also reported by Hoang et al. [ 76 ] for the srpay-drying encapsulation of Hibiscus sabdariffa extract using maltodextrin and Arabic gum. Figure 5 illustrates the particle size distribution of microparticles loaded with E. faecium EM03, averaging 10.48 ± 0.80 µm. The volume-weighted mean diameter (D[ 4 , 3 ]) was 11.47 ± 0.44 µm, while the surface area-weighted mean diameter (D[ 3 , 2 ]) was 5.64 ± 0.21 µm, resulting in a span of 1.61 ± 0.05 µm. D[ 4 , 3 ] highlights the presence of larger particles in the sample, with higher values indicating a greater contribution from these larger particles. In contrast, D[ 3 , 2 ] emphasizes smaller particles, which are particularly important for surface-dependent processes, as lower values correspond to finer particles that possess a larger total surface area. The Span, calculated as (D90 – D10) / D50, provides insight into the breadth of the particle size distribution; lower values signify a narrower distribution, whereas higher values indicate a broader one. Together, these parameters are crucial for characterizing average particle size and polydispersity [ 77 ]. From a technological perspective, larger particles may enhance probiotic protection during atomization, as bigger droplets tend to heat more slowly and lose water more gradually during spray drying, reducing thermal and dehydration stress on the cells. In this study, the span value (1.61) suggests a moderately broad yet acceptable distribution, which is commonly observed in polysaccharide-based spray-dried systems and is unlikely to impair powder flowability or application. In 2025, Fahrudin et al. [ 78 ] reported an average particle size of approximately 5.2 µm, with D[ 4 , 3 ] measuring 4.45 µm for microparticles produced from Arabic gum and vegetable extracts. The smaller particles reported by Fahrudin et al. [ 78 ] may also be related to the use of Arabic gum as the sole wall material. In contrast, the present formulation combined 20% Arabic gum with 5% inulin, which increases the feed viscosity and typically leads to the formation of larger droplets during atomization. This effect has been widely described in spray-dried polysaccharide systems, where higher viscosity promotes larger particle sizes and broader distributions. Also, this variation in values may stem from differences in equipment and drying parameters, which directly affect the size and morphology of the resulting powder. The resistance of both free and encapsulated bacteria to simulated gastrointestinal conditions was assessed, with results illustrated in Fig. 6 . The population of free bacteria began to decline after 90 minutes, along with the conclusion of the gastric phase. In contrast, the population of microencapsulated bacteria started to decrease after 45 minutes, during the mid-gastric phase. Both types of situations exhibited remarkable survival rates, with 86% surviving by the end of the assay. These findings suggest that the microencapsulation of E. faecium EM03 could serve as an effective technological approach to enhance bacterial survival throughout a product's shelf life, thereby facilitating its industrial application, as powders are generally easier to handle. The evaluations conducted by Paula et al. [ 79 ] demonstrated that Arabi gum significantly enhanced the survival of L. plantarum strains, with viability rates reaching 80% for microencapsulated cells compared to their free counterparts. However, a decrease in viability during the gastric phase was noted, attributable to the acidic conditions. Additionally, the favorable outcomes associated with the incorporation of inulin into the encapsulating matrix align with the findings of Ahmadi et al. [ 80 ] and Meral et al. [ 81 ]. These studies also observed an increased viability of probiotic bacteria, specifically L. acidophilus LA-5 and L. rhamnosus , when these strains were encapsulated with inulin, offering superior protection compared to those encapsulated without this compound. Consequently, the combined use of Arabic gum and inulin as wall materials for encapsulation may provide enhanced protection for probiotic strains compared to the use of either material in isolation, as evidenced by this study and other research [ 82 ]. 3.4.1 Effect of storage temperature Microparticles were stored at temperatures of 4°C and 25°C for up to 60 days, and bacterial counts were performed on samples taken at 0, 15, 30, 45, and 60 days. In addition, analyses of water activity, SEM, FT-IR and size distribution were conducted following the 60-day storage period. The resulting data is presented in Table 3 and Figs. 8 , 9 , and 10. Table 3 Bacterial population, water activity average size, volume-weighted mean diameter (D[ 4 , 3 ]), surface area-weighted mean diameter (D[ 3 , 2 ]) and span of microparticles loaded with Enterococcus faecium EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin. The microparticles were stored at 4°C and 25°C for up to 60 days. Different letters in a row indicate significant differences ( p < 0.05). Temperature Parameter 0 days 15 days 30 days 45 days 60 days 4°C Bacterial population (log CFU/g) 8.53 ± 0.05 a 8.62 ± 0.15 ab 8.93 ± 0.21 b 8.85 ± 0.22 a 8.52 ± 0.24 a Water activity 0.320 ± 0.02 a n.d. n.d. n.d. 0.345 ± 0.01 a Average size (µm) 10.48 ± 0.80 a n.d. n.d. n.d. 9.40 ± 0.80 a D[ 4 , 3 ] (µm) 11.47 ± 0.44 a n.d. n.d. n.d. 13.02 ± 0.22 b D[ 3 , 2 ] (µm) 5.64 ± 0.21 a n.d. n.d. n.d. 6.46 ± 0.10 b Span (µm) 1.61 ± 0.05 a n.d. n.d. n.d. 1.39 ± 0.00 b 25°C Bacterial population (log CFU/g) 8.53 ± 0.05 a 8.40 ± 0.09 a 7.95 ± 0.00 b 8.50 ± 0.05 a 8.09 ± 0.02 b Water activity 0.320 ± 0.02 a n.d. n.d. n.d. 0.467 ± 0.02 b Average size 10.48 ± 0.80 a n.d. n.d. n.d. 11.59 ± 0.92 a D[ 4 , 3 ] 11.47 ± 0.44 a n.d. n.d. n.d. 9.70 ± 0.70 a D[ 3 , 2 ] 5.64 ± 0.21 a n.d. n.d. n.d. 5.03 ± 0.30 a Span 1.61 ± 0.05 a n.d. n.d. n.d. 1.59 ± 0.02 a n.d.: not determined The data presented in Table 3 regarding the bacterial population suggests that the formulation stored at 4°C for 60 days successfully maintained its original bacterial count. In contrast, the formulation stored at 25°C for the same duration exhibited a slight decline in bacterial population compared to the initial count. This reduction can likely be attributed to higher temperatures, as 25°C is more favorable for bacterial metabolism than 4°C. Furthermore, it is noteworthy that the water activity (A w ) at 25°C increased, which may have further enhanced bacterial metabolism during storage. Overall, the stability observed during storage can also be attributed to the presence of prebiotics within the matrix, which contributed to the viability of the bacteria. These findings suggest that the synergistic effect of Arabic gum and inulin offers protection and enhances the durability of probiotics during storage. Studies utilizing inulin as encapsulating agent for L. acidophilus have reported positive effects on bacterial stability, achieving high viability rates of 7.72 log CFU/g compared to particles lacking inulin [ 80 ]. Similarly, Naseem et al. [ 74 ] highlighted the effectiveness of Arabic gum as a protective material against environmental factors, resulting in probiotic microparticles exhibiting higher survival rates and thermal stability. Additionally, findings from Yin et al. [ 83 ] indicated that encapsulation using Arabic gum remained effective when stored at 4°C for 16 weeks, showing only a reduction of less than 1.5 log CFU/g. However, a decline in viable cells was noted when exposed to 25°C, reflecting patterns similar to those observed in the present study. In conclusion, the data suggests that the combination of Arabic gum and inulin as encapsulating agents provides enhanced protection against external factors, with inulin also supporting the survival of probiotic bacteria due to its prebiotic properties. Figure 7 revealed that the morphological structures kept visuable the same for particles stored at 4 and 25°C, and also very similar to the particles presented in Fig. 3 immediately after their production. Figure 8 also indicates that there was no change in the chemical interactions between the components of microparticles, neither a change in the spectra pattern. Similarly, Fig. 9 indicates the same pattern of size differences for particles store at both conditions, although Table 4 showed an increase on D[ 4 , 3 ] and D[ 3 , 2 ] values for particles stored at 4°C, which may be attributed to the low temperature of storage. Taken together, these results suggests that microparticles loaded with Enterococcus faecium EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin, may be successfully stored at 4°C for up to 60 days without compromising microbiological and physical parameters. 4. Conclusions E. faecium EM03 exhibits significant technological potential for protecting against foodborne pathogens, as evidenced by the presence of genes responsible for the production bacteriocins, its strong antilisterial activity, and its probiotic properties, which are demonstrated by its ability to withstand conditions simulating passage through the gastrointestinal tract. The safety assessment indicated no resistance to clinically important antibiotics and absence of genes for the production of virulence factors and biogenic amines. Additionally, the application of the spray-drying encapsulation technique utilizing the Arabic gum–inulin complex as a wall matrix has proven effective in enhancing the strain's viability and stability during storage, as well as protecting it from adverse conditions and enzymatic action. Collectively, these findings suggest that E. faecium EM03 possesses broad functional potential that remains largely unexplored, particularly concerning the enterocins it produces and its probiotic effects, including bile salt hydrolase activity. Therefore, future studies are crucial to deepen our understanding of its functionalities, facilitate the discovery of new applications, and expand the scientific knowledge surrounding this species. Declarations The authors declare no competing interests. The data underlying this article will be shared on reasonable request to the corresponding author. Acknowledgments We acknowledge Dennis Coelho Cruz for assistance with SEM image acquisition and MCTI/FINEP/FNDCT/CT-INFRA facilities for instrumental analyses. The research leading to these results received funding from CNPq (National Council for Scientific and Technological Development: 403613/2023-0), and FAPESB (Foundation for Research Support of the State of Bahia: BioproFar-BA PIE0001/2024; PPF0004/2023; PIE0006/2022; PPF0017/2021). Author Contributions Statement E.S.O.M. conducted microbiological, genetic, and physical analyses, drafted the manuscript, and contributed to its revision. G.E.N.O., B.P.S. and M.P.S.P. performed microparticle analyses and contributed to manuscript preparation. I.R. executed the genetic sequencing analyses. E.C.P.D.M. supervised the genetic sequencing analyses and contributed to both writing and revising the manuscript. I.A.G. also contributed to the writing and revision of the manuscript. M.H.F.K. carried out and supervised the genotyping analyses, and contributed to the manuscript writing and revision. F.L.T. managed the project, provided funding, and contributed to the writing and revision of the manuscript. References FAO, WHO (2002) Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food Das TK, Pradhan S, Chakrabarti S et al (2022) Current status of probiotic and related health benefits. Appl Food Res 2:100185. https://doi.org/10.1016/j.afres.2022.100185 Liao W, Chen C, Wen T, Zhao Q (2021) Probiotics for the Prevention of Antibiotic-associated Diarrhea in Adults. J Clin Gastroenterol 55:469–480. https://doi.org/10.1097/MCG.0000000000001464 Zheng J, Wittouck S, Salvetti E et al (2020) A taxonomic note on the genus Lactobacillus : Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol 70:2782–2858. https://doi.org/10.1099/ijsem.0.004107 Li J, Wang J, Wang M et al (2023) Bifidobacterium : a probiotic for the prevention and treatment of depression. Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1174800 Mulaw G, Tessema TS, Muleta D, Tesfaye A (2019) In Vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from Some Traditionally Fermented Ethiopian Food Products. Int J Microbiol 2019:1–11. https://doi.org/10.1155/2019/7179514 Giraffa G (2002) Enterococci from foods. FEMS Microbiol Rev 26:163–171. https://doi.org/10.1016/S0168-6445(02)00094-3 Franz CMAP, Van Belkum MJ, Holzapfel WH et al (2007) Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31:293–310. https://doi.org/10.1111/j.1574-6976.2007.00064.x Rotta IS, Rodrigues WF, Santos CTB et al (2022) Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants. Microb Pathog 171:105715. https://doi.org/10.1016/j.micpath.2022.105715 Gundog DA, Onmaz NE, Gungor C et al (2025) Enterococcus faecalis and E. faecium in dairy production line: Antibiotic resistance profile and virulence characteristics. Int Dairy J 165:106209. https://doi.org/10.1016/j.idairyj.2025.106209 Gomes BC, Esteves CT, Palazzo ICV et al (2008) Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods. Food Microbiol 25:668–675. https://doi.org/10.1016/j.fm.2008.03.008 Turner S, Pryer KM, Miao VPW, Palmer JD (1999) Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis1. J Eukaryot Microbiol 46:327–338. https://doi.org/10.1111/j.1550-7408.1999.tb04612.x Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175 Vankerckhoven V, Van Autgaerden T, Vael C et al (2004) Development of a Multiplex PCR for the Detection of asa1 , gelE , cylA , esp , and hyl Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of Enterococcus faecium . J Clin Microbiol 42:4473–4479. https://doi.org/10.1128/JCM.42.10.4473-4479.2004 Moraes PM, Perin LM, Todorov SD et al (2012) Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese. J Appl Microbiol 113:318–328. https://doi.org/10.1111/j.1365-2672.2012.05341.x Tulini FL, Bíscola V, Choiset Y et al (2015) Evaluation of the proteolytic activity of Enterococcus faecalis FT132 and Lactobacillus paracasei FT700, isolated from dairy products in Brazil, using milk proteins as substrates. Eur Food Res Technol 241. https://doi.org/10.1007/s00217-015-2470-6 Omar N, Ben, Castro A, Lucas R et al (2004) Functional and Safety Aspects of Enterococci Isolated from Different Spanish Foods. Syst Appl Microbiol 27:118–130. https://doi.org/10.1078/0723-2020-00248 Eaton TJ, Gasson MJ (2001) Molecular Screening of Enterococcus Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates. Appl Environ Microbiol 67:1628–1635. https://doi.org/10.1128/AEM.67.4.1628-1635.2001 Rivas P, Alonso J, Moya J et al (2005) The Impact of Hospital-Acquired Infections on the Microbial Etiology and Prognosis of Late-Onset Prosthetic Valve Endocarditis. Chest 128:764–771. https://doi.org/10.1378/chest.128.2.764 Albano H, Todorov SD, van Reenen CA et al (2007) Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from Alheira, a fermented sausage traditionally produced in Portugal. Int J Food Microbiol 116:239–247. https://doi.org/10.1016/j.ijfoodmicro.2007.01.011 Hyink O, Balakrishnan M, Tagg JR (2005) Streptococcus rattus strain BHT produces both a class I two-component lantibiotic and a class II bacteriocin. FEMS Microbiol Lett 252:235–241. https://doi.org/10.1016/j.femsle.2005.09.003 Wirawan RE, Klesse NA, Jack RW, Tagg JR (2006) Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis . Appl Environ Microbiol 72:1148–1156. https://doi.org/10.1128/AEM.72.2.1148-1156.2006 Du Toit M, Franz CMAP, Dicks LMT, Holzapfel WH (2000) Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces. J Appl Microbiol 88:482–494. https://doi.org/10.1046/j.1365-2672.2000.00986.x Dutka-Malen S, Evers S, Courvalin P (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24–27. https://doi.org/10.1128/jcm.33.1.24-27.1995 Wang C-Y, Lin P-R, Ng C-C, Shyu Y-T (2010) Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage. Anaerobe 16:578–585. https://doi.org/10.1016/j.anaerobe.2010.10.003 Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially probiotic Lactobacillus species. J Food Prot 61:1636–1643. https://doi.org/10.4315/0362-028X-61.12.1636 Lewus CB, Kaiser A, Montville TJ (1991) Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Appl Environ Microbiol 57:1683–1688. https://doi.org/10.1128/aem.57.6.1683-1688.1991 Tulini FL, Winkelströter LK, De Martinis ECP (2013) Identification and evaluation of the probiotic potential of Lactobacillus paraplantarum FT259, a bacteriocinogenic strain isolated from Brazilian semi-hard artisanal cheese. Anaerobe 22. https://doi.org/10.1016/j.anaerobe.2013.06.006 Hoben HJ, Somasegaran P (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl Environ Microbiol 44:1246–1247. https://doi.org/10.1128/aem.44.5.1246-1247.1982 Brodkorb A, Egger L, Alminger M et al (2019) INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat Protoc 14:991–1014. https://doi.org/10.1038/s41596-018-0119-1 Kim Y, Choi S-I, Jeong Y, Kang C-H (2022) Evaluation of safety and probiotic potential of Enterococcus faecalis MG5206 and Enterococcus faecium MG5232 isolated from Kimchi, a Korean fermented cabbage. Microorganisms 10:2070. https://doi.org/10.3390/microorganisms10102070 Chajęcka-Wierzchowska W, Zadernowska A, Łaniewska-Trokenheim Ł (2017) Virulence factors of Enterococcus spp. presented in food. LWT 75:670–676. https://doi.org/10.1016/j.lwt.2016.10.026 Fuka MM, Maksimovic AZ, Tanuwidjaja I et al (2017) Characterization of enterococcal community isolated from an artisan Istrian raw milk cheese: biotechnological and safety aspects. Food Technol Biotechnol 55. https://doi.org/10.17113/ftb.55.03.17.5118 Tsanasidou C, Asimakoula S, Sameli N et al (2021) Safety evaluation, biogenic amine formation, and enzymatic activity profiles of autochthonous enterocin-producing greek cheese isolates of the Enterococcus faecium/durans Group. Microorganisms 9:777. https://doi.org/10.3390/microorganisms9040777 Kiruthiga A, Padmavathy K, Shabana P et al (2020) Improved detection of esp , hyl , asa 1, gel E, cyl A virulence genes among clinical isolates of Enterococci. BMC Res Notes 13:170. https://doi.org/10.1186/s13104-020-05018-0 Ennahar S (2000) Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol Rev 24:85–106. https://doi.org/10.1016/S0168-6445(99)00031-5 Favaro L, Basaglia M, Casella S et al (2014) Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese. Food Microbiol 38:228–239. https://doi.org/10.1016/j.fm.2013.09.008 Valledor SJD, Dioso CM, Bucheli JEV et al (2022) Characterization and safety evaluation of two beneficial, enterocin-producing Enterococcus faecium strains isolated from kimchi, a Korean fermented cabbage. Food Microbiol 102:103886. https://doi.org/10.1016/j.fm.2021.103886 Trościańczyk A, Nowakiewicz A, Tracz AM, Bochniarz M (2025) Evaluation of the activity and molecular characterisation of bacteriocins produced by E. faecium and E. faecalis isolated from different hosts against public health-threating pathogens. Microb Pathog 202:107432. https://doi.org/10.1016/j.micpath.2025.107432 Mancini L, Carbone S, Calabrese FM et al (2025) Isolation and characterization from raw milk of Enterocin B-producing Enterococcus faecium : A potential dairy bio-preservative agent. Appl Food Res 5:100857. https://doi.org/10.1016/j.afres.2025.100857 Sonsa-Ard N, Rodtong S, Chikindas ML, Yongsawatdigul J (2015) Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe. Food Control 54:308–316. https://doi.org/10.1016/j.foodcont.2015.02.010 Asaduzzaman SM, Sonomoto K (2009) Lantibiotics: Diverse activities and unique modes of action. J Biosci Bioeng 107:475–487. https://doi.org/10.1016/j.jbiosc.2009.01.003 Nagao J (2009) Properties and applications of lantibiotics, a class of bacteriocins produced by Gram-positive bacteria. J Oral Biosci 51:158–164. https://doi.org/10.1016/S1349-0079(09)80024-8 Franz CMAP, Huch M, Abriouel H et al (2011) Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151:125–140. https://doi.org/10.1016/j.ijfoodmicro.2011.08.014 Çetin B, Aktaş H (2024) Monitoring probiotic properties and safety evaluation of antilisterial Enterococcus faecium strains with cholesterol-lowering potential from raw Cow’s milk. Food Biosci 61:104532. https://doi.org/10.1016/j.fbio.2024.104532 Amidi-Fazli N, Hanifian S (2022) Biodiversity, antibiotic resistance and virulence traits of Enterococcus species in artisanal dairy products. Int Dairy J 129:105287. https://doi.org/10.1016/j.idairyj.2021.105287 Özdemir R, Tuncer Y (2020) Detection of antibiotic resistance profiles and aminoglycoside-modifying enzyme (AME) genes in high-level aminoglycoside-resistant (HLAR) Enterococci isolated from raw milk and traditional cheeses in Turkey. Mol Biol Rep 47:1703–1712. https://doi.org/10.1007/s11033-020-05262-4 Arias CA, Murray BE (2012) The rise of the Enterococcus : beyond vancomycin resistance. Nat Rev Microbiol 10:266–278. https://doi.org/10.1038/nrmicro2761 Cariolato D, Andrighetto C, Lombardi A (2008) Occurrence of virulence factors and antibiotic resistances in Enterococcus faecalis and Enterococcus faecium collected from dairy and human samples in North Italy. Food Control 19:886–892. https://doi.org/10.1016/j.foodcont.2007.08.019 Costa Y, Galimand M, Leclercq R et al (1993) Characterization of the chromosomal aac(6’)-Ii gene specific for Enterococcus faecium. Antimicrob Agents Chemother 37:1896–1903. https://doi.org/10.1128/AAC.37.9.1896 Charyyev MG, Tuncer BÖ, Kankaya DA, Tuncer Y (2019) Bacteriocinogenic properties and safety evaluation of Enterococcus faecium YT52 isolated from boza, a traditional cereal based fermented beverage. J Consumer Prot Food Saf 14:41–53. https://doi.org/10.1007/s00003-019-01213-9 Nami Y, Bakhshayesh RV, Jalaly HM et al (2019) Probiotic properties of Enterococcus isolated from artisanal dairy products. Front Microbiol 10. https://doi.org/10.3389/fmicb.2019.00300 Furlaneto-Maia L, Ramalho R, Rocha KR, Furlaneto MC (2020) Antimicrobial activity of enterocins against Listeria sp. and other food spoilage bacteria. Biotechnol Lett 42:797–806. https://doi.org/10.1007/s10529-020-02810-7 Du R, Ping W, Ge J (2022) Purification, characterization and mechanism of action of enterocin HDX-2, a novel class IIa bacteriocin produced by Enterococcus faecium HDX-2. LWT 153:112451. https://doi.org/10.1016/j.lwt.2021.112451 Zhou C, Chang X, Zou Y et al (2024) The mechanism of Enterococcus faecium on the virulence of Listeria monocytogenes during the storage of fermented sausages by whole genome analysis. Int J Food Microbiol 422:110826. https://doi.org/10.1016/j.ijfoodmicro.2024.110826 Schittler L, Perin LM, Marques JL et al (2019) Isolation of Enterococcus faecium , characterization of its antimicrobial metabolites and viability in probiotic Minas Frescal cheese. J Food Sci Technol 56:5128–5137. https://doi.org/10.1007/s13197-019-03985-2 Popović N, Stevanović D, Radojević D et al (2023) Insight into the postbiotic potential of the autochthonous bacteriocin-producing Enterococcus faecium BGZLM1-5 in the reduction in the abundance of Listeria monocytogenes ATCC19111 in a milk model. Microorganisms 11:2844. https://doi.org/10.3390/microorganisms11122844 Boke H, Aslim B, Alp G (2010) The role of resistance to bile salts and acid tolerance of exopolysaccharides (EPSS) produced by yogurt starter bacteria. Arch Biol Sci 62:323–328. https://doi.org/10.2298/ABS1002323B Gupta SBR, Sraboni FS, Naznin T et al (2025) Harnessing Enterococcus faecium WFD-128 from yogurt fermentation: Unveiling probiotic attributes and targeted inhibition of Shigella sonnei diarrheal pathogenesis. Microb Pathog 204:107561. https://doi.org/10.1016/j.micpath.2025.107561 Ankaiah D, Esakkiraj P, Perumal V et al (2017) Probiotic characterization of Enterococcus faecium por1: Cloning, over expression of Enterocin-A and evaluation of antibacterial, anti-cancer properties. J Funct Foods 38:280–292. https://doi.org/10.1016/j.jff.2017.09.034 Xiao J, Chen C, Fu Z et al (2024) Assessment of the safety and probiotic properties of Enterococcus faecium B13 isolated from fermented Chili. Microorganisms 12:994. https://doi.org/10.3390/microorganisms12050994 Bhardwaj A, Gupta H, Kapila S et al (2010) Safety assessment and evaluation of probiotic potential of bacteriocinogenic Enterococcus faecium KH 24 strain under in vitro and in vivo conditions. Int J Food Microbiol 141:156–164. https://doi.org/10.1016/j.ijfoodmicro.2010.05.001 Abedini R, Zaghari G, Jabbari L et al (2023) A potential probiotic Enterococcus faecium isolated from camel rumen, fatty acids biotransformation, antilisteria activity and safety assessment. Int Dairy J 145:105706. https://doi.org/10.1016/j.idairyj.2023.105706 Guan N, Liu L (2020) Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biotechnol 104:51–65. https://doi.org/10.1007/s00253-019-10226-1 Singh J, Metrani R, Shivanagoudra SR et al (2019) Review on bile acids: Effects of the gut microbiome, interactions with dietary fiber, and alterations in the bioaccessibility of bioactive compounds. J Agric Food Chem 67:9124–9138. https://doi.org/10.1021/acs.jafc.8b07306 Tilwani YM, Lakra AK, Domdi L et al (2022) Characterization of potential probiotic bacteria Enterococcus faecium MC-5 isolated from the gut content of Cyprinus carpio specularis. Microb Pathog 172:105783. https://doi.org/10.1016/j.micpath.2022.105783 Almeida KV, Zanetti VC, Camelo-Silva C et al (2024) Powdered water kefir: Effect of spray drying and lyophilization on physical, physicochemical, and microbiological properties. Food Chem Adv 5:100759. https://doi.org/10.1016/j.focha.2024.100759 Barbosa J, Borges S, Amorim M et al (2015) Comparison of spray drying, freeze drying and convective hot air drying for the production of a probiotic orange powder. J Funct Foods 17:340–351. https://doi.org/10.1016/j.jff.2015.06.001 Chang Y, Lin T, Zhu M, Yu S (2025) Complexation of Inulin with Proteins, Polysaccharides and Polyphenols: Their Interactions, Applications and Health Benefits. Food Reviews Int 41:1933–1947. https://doi.org/10.1080/87559129.2025.2456544 Kingwatee N, Apichartsrangkoon A, Chaikham P et al (2015) Spray drying Lactobacillus casei 01 in lychee juice varied carrier materials. LWT - Food Sci Technol 62:847–853. https://doi.org/10.1016/j.lwt.2014.12.007 Huang S, Vignolles M-L, Chen XD et al (2017) Spray drying of probiotics and other food-grade bacteria: A review. Trends Food Sci Technol 63:1–17. https://doi.org/10.1016/j.tifs.2017.02.007 Ramírez-Damián M, Garfias-Noguez C, Bermúdez-Humarán LG, Sánchez-Pardo ME (2025) Synbiotic microencapsulation of Lactobacillus strains from mexican fermented beverages for enhanced probiotic functionality. Molecules 30:1185. https://doi.org/10.3390/molecules30051185 Reyes V, Chotiko A, Chouljenko A, Sathivel S (2018) Viability of Lactobacillus acidophilus NRRL B-4495 encapsulated with high maize starch, maltodextrin, and gum arabic. LWT 96:642–647. https://doi.org/10.1016/j.lwt.2018.06.017 Naseem Z, Mir SA, Wani SM et al (2025) Investigating gum arabic and soy protein isolate as wall material for encapsulation of five strains of Lactobacillus . Int J Biol Macromol 298:140083. https://doi.org/10.1016/j.ijbiomac.2025.140083 Karrar E, Mahdi AA, Sheth S et al (2021) Effect of maltodextrin combination with gum arabic and whey protein isolate on the microencapsulation of gurum seed oil using a spray-drying method. Int J Biol Macromol 171:208–216. https://doi.org/10.1016/j.ijbiomac.2020.12.045 Hoang NTN, Nguyen NNK, Nguyen LTK et al (2024) Research on optimization of spray drying conditions, characteristics of anthocyanins extracted from Hibiscus sabdariffa L. flower, and application to marshmallows. Food Sci Nutr 12:2003–2015. https://doi.org/10.1002/fsn3.3898 Konstanty J, Tyrala D (2024) Particle sizing and surface area measurements: A comparative assessment of commercial air permeability and laser light diffraction instruments. Appl Sci 14:4802. https://doi.org/10.3390/app14114802 Fahrudin FI, Phongthai S, Intipunya P (2025) Enhancing stability of Boesenbergia rotunda bioactive compounds: Microencapsulation via spray-drying and its physicochemical evaluation. Foods 14:2699. https://doi.org/10.3390/foods14152699 Paula DA, Martins EMF, Costa NA et al (2019) Use of gelatin and gum arabic for microencapsulation of probiotic cells from Lactobacillus plantarum by a dual process combining double emulsification followed by complex coacervation. Int J Biol Macromol 133:722–731. https://doi.org/10.1016/j.ijbiomac.2019.04.110 Ahmadi M, Khajeh F, Sohrabi S et al (2025) Spray-dried probiotic microcapsules with calcium cross-linked oxidized starch and inulin. Carbohydr Polym Technol Appl 10:100760. https://doi.org/10.1016/j.carpta.2025.100760 Meral HD, Özcan FŞ, Özcan N et al (2024) Determination of prebiotic activity and probiotic encapsulation ability of inulin type fructans obtained from Inula helenium roots. J Food Sci 89:5335–5349. https://doi.org/10.1111/1750-3841.17261 D’Amico V, Siepmann F, Siepmann J et al (2026) Microencapsulation of the probiotic Bifidobacterium longum by spray-drying: Formulation and process optimisation. J Drug Deliv Sci Technol 115:107670. https://doi.org/10.1016/j.jddst.2025.107670 Yin M, Chen M, Yuan Y et al (2024) Encapsulation of Lactobacillus rhamnosus GG in whey protein isolate-shortening oil and gum Arabic by complex coacervation: Enhanced the viability of probiotics during spray drying and storage. Food Hydrocoll 146:109252. https://doi.org/10.1016/j.foodhyd.2023.109252 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviews received at journal 20 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviews received at journal 27 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers invited by journal 09 Dec, 2025 Editor assigned by journal 07 Dec, 2025 Submission checks completed at journal 07 Dec, 2025 First submitted to journal 06 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8297099","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559275397,"identity":"f4de0755-a0dd-4113-bf2c-26b18ff9e81c","order_by":0,"name":"Emanuela Stefany Oliveira Maciel","email":"","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":false,"prefix":"","firstName":"Emanuela","middleName":"Stefany Oliveira","lastName":"Maciel","suffix":""},{"id":559275398,"identity":"716d57ff-3e73-411a-9deb-597043d99cac","order_by":1,"name":"Giovanni Eiji do Nascimento Ozaki","email":"","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":false,"prefix":"","firstName":"Giovanni","middleName":"Eiji do Nascimento","lastName":"Ozaki","suffix":""},{"id":559275399,"identity":"bbef7161-4af9-412d-a30e-26b5996b72dd","order_by":2,"name":"Bruna Peregrino Souza","email":"","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":false,"prefix":"","firstName":"Bruna","middleName":"Peregrino","lastName":"Souza","suffix":""},{"id":559275400,"identity":"407ea5c5-7265-4861-a7c6-f95275e2fbf6","order_by":3,"name":"Marluci Palazzolli Silva Padilha","email":"","orcid":"","institution":"State University of Campinas","correspondingAuthor":false,"prefix":"","firstName":"Marluci","middleName":"Palazzolli Silva","lastName":"Padilha","suffix":""},{"id":559275401,"identity":"c867c532-7cc8-41ba-873b-7edf4c9e3942","order_by":4,"name":"Inaie Reinecke","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Inaie","middleName":"","lastName":"Reinecke","suffix":""},{"id":559275402,"identity":"1e182f60-fd0f-4ec2-a7c3-12254a2f825b","order_by":5,"name":"Elaine Cristina Pereira De Martinis","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Elaine","middleName":"Cristina Pereira","lastName":"De Martinis","suffix":""},{"id":559275403,"identity":"677979dd-7b3a-4376-8dfa-833acfa01a1c","order_by":6,"name":"Izabela Alves Gomes","email":"","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"Alves","lastName":"Gomes","suffix":""},{"id":559275405,"identity":"145b75df-b5c4-4702-b269-c6f69b2ecdbd","order_by":7,"name":"Mary Hellen Fabres Klein","email":"","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"Hellen Fabres","lastName":"Klein","suffix":""},{"id":559275406,"identity":"6ab1a8cd-5673-4091-8e3b-2978737df08a","order_by":8,"name":"Fabricio Luiz Tulini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBAC9gYeBoYEBgkQm/EAkCHHwHyADchhZoAIYgKeAzAtQHUgLcYMbAlEaAEDkBYgldhAUAv72WMfHtRYyDHI9xgceLjDIn3DMeZjDxgqrBMbpHsfYNXCk5c8I+EYyD08BgcSz0jkbjjGlm7AcCY9sUHmuAE2LfYMOcZAx0gA3QPS0gbUcr/HTIKx7TBQMA27w/jfoGpJNzjGA9TyD48WCTRbEiBaGvBpeZfMAPILG1taAUiL4UyQXxKOpRu3yRzD4bDcw4w/aurk+JkPb3z4s61Ong8UYh9qrGX7pduwaoEDNhReAobIKBgFo2AUjAJSAACu+lh5HZNKIAAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Western Bahia","correspondingAuthor":true,"prefix":"","firstName":"Fabricio","middleName":"Luiz","lastName":"Tulini","suffix":""}],"badges":[],"createdAt":"2025-12-06 23:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8297099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8297099/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98432268,"identity":"c8b29fa4-3fbc-48f0-9747-26d88dedbd71","added_by":"auto","created_at":"2025-12-17 16:49:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3679008,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptEfaeciumEM03PAPwithoutMendeley.docx","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/bdcc2a890f601cd231d1a0dd.docx"},{"id":98211032,"identity":"3cbc82be-5563-41df-9833-32649915c341","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10648,"visible":true,"origin":"","legend":"","description":"","filename":"b09abceed9834db183ea7593f6738428.json","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/ea61f59f9b28270b791e374d.json"},{"id":98211041,"identity":"bb714c7a-74cd-4d74-ba89-ca2789438f7e","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":228926,"visible":true,"origin":"","legend":"","description":"","filename":"b09abceed9834db183ea7593f67384281enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/e370ad2f0c686e078aef5e41.xml"},{"id":98211035,"identity":"a662fc7d-8f59-4318-a94c-142edc3d711c","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":248316,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/8671127d91baebf7dd5c3f9f.png"},{"id":98622963,"identity":"cf72f636-0405-47d0-8fa5-3c9d085908fc","added_by":"auto","created_at":"2025-12-19 17:03:47","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87691,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/adcb8024e3cde6909af0a935.png"},{"id":98431612,"identity":"ba2f8317-80b5-4086-a353-bb84f0b00e89","added_by":"auto","created_at":"2025-12-17 16:48:02","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41981,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/8a7c7bef72252d4197dcc47b.png"},{"id":98211044,"identity":"209458ef-da61-4c58-8b8a-e5e02b36d062","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92817,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/3238570df8a51c7adb8ef847.jpeg"},{"id":98211036,"identity":"c3b5a4d8-6564-4a72-91d6-eda1be4f4aee","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78367,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/3d5551acfb5396330ce1268a.jpeg"},{"id":98432286,"identity":"e8ae7040-bffd-40ad-8d11-e545d4bdc02a","added_by":"auto","created_at":"2025-12-17 16:49:20","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":70242,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/b78378d61b17dc3559a9745b.png"},{"id":98433371,"identity":"bac3c1ec-8ccf-45ea-953d-adb9ff71e615","added_by":"auto","created_at":"2025-12-17 16:50:41","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22781,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/9447f157d1793f3332d972ae.png"},{"id":98433329,"identity":"5c2ff898-d741-47b2-ba1f-7e63181c436a","added_by":"auto","created_at":"2025-12-17 16:50:38","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63443,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/80832084bcb49e9ff9130595.png"},{"id":98431575,"identity":"d33781f0-a93e-4c5b-95ec-fd809cf5c5d7","added_by":"auto","created_at":"2025-12-17 16:47:58","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1801219,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/fd084c3ed7b06cd043dedb66.jpeg"},{"id":98432287,"identity":"756b84f2-329e-4540-bec6-5e98b40d2ac1","added_by":"auto","created_at":"2025-12-17 16:49:20","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79367,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/202f2fd6c647bfefe2b93925.png"},{"id":98211048,"identity":"d989f98a-df0d-47c1-90e6-0a08b1b9e0bb","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24390,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/4acd9d1f0842a7ab7ffec225.png"},{"id":98211051,"identity":"2b515bd0-8048-497c-b1bf-86e0b8709e24","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15328,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/268a242a50c02b59b9eba632.png"},{"id":98211049,"identity":"74854580-41d3-426b-acc4-ec650807bb6a","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9349,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/e1a1b5ac0f78913ec909711b.png"},{"id":98211056,"identity":"5a2e82c0-f994-4888-9d8f-62d1b6436ab5","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":404759,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/4b941f6294b822663ff55563.png"},{"id":98211055,"identity":"653dcdf6-dd9b-4fb7-a04a-265f85340b60","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":385605,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/16ecdd2a9b7055909e131ce6.png"},{"id":98433545,"identity":"5d87dcdd-2fb9-40f4-97a9-e5b3cd9ee52b","added_by":"auto","created_at":"2025-12-17 16:50:53","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23174,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/7b86448555872bd35ab351cf.png"},{"id":98211062,"identity":"d8a6b372-700e-46ad-a89a-a5c16c372ac6","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8111,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/24ab2790e146754d34a936b5.png"},{"id":98211060,"identity":"33647ad4-9cec-4f16-9e44-6e0689a4e012","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17622,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/ab3d9e799be10bd7caef1517.png"},{"id":98211059,"identity":"3f53f7b8-19c2-4466-a27f-3e89bbe554cb","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2653081,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/16b71284992c84b24326c30a.png"},{"id":98211057,"identity":"b2a4030d-2b6e-4c44-b774-65787cfad2e8","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22690,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/03d3c00c2097dfbafa5231e2.png"},{"id":98211063,"identity":"260cde29-0434-4629-a9ee-18a3e54ea2fe","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226317,"visible":true,"origin":"","legend":"","description":"","filename":"b09abceed9834db183ea7593f67384281structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/35a672bfb1cf829cc9345cc0.xml"},{"id":98211064,"identity":"c64d6536-9801-4482-a222-7fe0562fe039","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":246317,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/27de7881959513811d64a1d5.html"},{"id":98211030,"identity":"b60978fb-9655-4f5b-81b7-61c45e0e5e79","added_by":"auto","created_at":"2025-12-15 09:36:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88909,"visible":true,"origin":"","legend":"\u003cp\u003eSpot-on-the-lawn assay demonstrating the inhibition of \u003cem\u003eListeria monocytogenes\u003c/em\u003e by the production of bacteriocins from \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03. A – negative control (water); E – Enzyme α-Chymotrypsin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/af47158bea5f000ab11b7ac1.png"},{"id":98433126,"identity":"0e3ca793-6556-46d4-bd48-568a2ee3767e","added_by":"auto","created_at":"2025-12-17 16:50:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27594,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eEnterococcus faecium\u003c/em\u003eEM03 under conditions simulating the gastrointestinal environment. For that, it was used: (i) BHI broth was adjusted to pH 2.0, 2.5, or 3.5 with acid; (ii) BHI broth containing 0.3% w/v bile salts; (iii) 0.9% w/v NaCl solution containing 3 g/L pepsin and adjusted to pH 2.0; (iv) unmodified BHI broth as a control. Significant variations within a same condition (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) are indicated by “ * ”.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/8a4f0a3c47d657003c406b91.png"},{"id":98431971,"identity":"b99efce4-6e20-49d2-b93a-35661acb8471","added_by":"auto","created_at":"2025-12-17 16:48:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301597,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin. (a) Microparticles at 2,000× magnification; (b) Microparticles at 5,000× magnification.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/25ccb7d31ead428f7e28bd6d.png"},{"id":98431312,"identity":"e4f9a813-0a74-4159-b1d7-a40ae7b70e57","added_by":"auto","created_at":"2025-12-17 16:47:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74266,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FT-IR) spectra in the 4000 to 400 cm\u003csup\u003e-1\u003c/sup\u003e regions of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin. Wall materials were also analyzed in the same conditions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/b312e3e368ca1e1ae8b60389.png"},{"id":98211033,"identity":"24c45e1a-a620-4a25-bf91-669edf79b392","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26948,"visible":true,"origin":"","legend":"\u003cp\u003eSize distribution of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin, expressed as a volume-based particle size distribution (%).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/e53ea62aa7b1989ed26a8c1d.png"},{"id":98211038,"identity":"5502d505-b46f-4aed-ae8f-0b80baff13f4","added_by":"auto","created_at":"2025-12-15 09:36:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49814,"visible":true,"origin":"","legend":"\u003cp\u003eResistance of both free bacteria and microencapsulated Enterococcus faecium EM03 to simulated gastrointestinal conditions. The microencapsulation was achieved through spray-drying a formulation consisting of 20% Arabic gum and 5% inulin. In this figure, “G” denotes simulated gastric fluid (SGF), while “I” indicates simulated intestinal fluid (SIF). Bars marked with “*” have significant differences in comparison to the control at T0G (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/ce54eddca9ecab9f6172a83d.png"},{"id":98433123,"identity":"15ed0007-1e6b-4d72-be68-291f73ea9be3","added_by":"auto","created_at":"2025-12-17 16:50:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":587629,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin. (a) Microparticles stored at 4°C for 60 days at 2,000× magnification; (b) Microparticles stored at 4°C for 60 days at 5,000× magnification; (c) Microparticles stored at 25°C for 60 days at 2,000× magnification; (d) Microparticles stored at 25°C for 60 days at 5,000× magnification.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/96f329706bfae536e328ee95.png"},{"id":98432297,"identity":"e28baa34-4700-4b90-bc2e-6d52514475df","added_by":"auto","created_at":"2025-12-17 16:49:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":73344,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FT-IR) spectra in the 4000 to 400 cm\u003csup\u003e-1\u003c/sup\u003e regions of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin, stored at 4°C and 25°C for up to 60 days.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/e04c030619f581c334e24ad1.png"},{"id":98431989,"identity":"4d289eee-303e-456a-82a0-e1ed443e6fee","added_by":"auto","created_at":"2025-12-17 16:48:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":64981,"visible":true,"origin":"","legend":"\u003cp\u003eSize distribution of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin, stored at 4°C and 25°C for up to 60 days.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/4bcf256271d2c0bca33b4df5.png"},{"id":98631268,"identity":"6b3a62da-5f9a-437c-8b7a-fb6ef1dcfc2d","added_by":"auto","created_at":"2025-12-19 17:19:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2770120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8297099/v1/e988fa98-01e6-404f-ae75-4724ac819ae1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Probiotic and technological potential of Enterococcus faecium EM03, isolated from Brazilian semi-hard cheese","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eProbiotics are defined by the the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] as \u0026ldquo;live microorganisms which, when administered in adequate amounts, confer a health benefit on the host\u0026rdquo;. Probiotic bacteria comprise mainly lactic acid bacteria (LAB), which are non-sporulating, Gram-positive, catalase-negative cocci, coccobacilli or rods. LAB can produce lactic acid via carbohydrate metabolism besides other antimicrobial compounds synthesized by diverse pathways. In addition, LAB may offer a protective barrier for the intestinal mucosa and promote several health benefits, including modulation of the immune system [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The beneficial effects of LAB have been widely investigated but several mechanisms of action remain inconclusive [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and \u003cem\u003eEnterococcus\u003c/em\u003e are the main genera of LAB [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which are naturally found in foods and as commensals in the gastrointestinal tract of humans and animals. The genus \u003cem\u003eEnterococcus\u003c/em\u003e includes a total of 19 cataloged species, according to chemotaxonomic and phylogenetic studies, with \u003cem\u003eEnterococcus faecium\u003c/em\u003e being one of the most extensively studied species [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e is able to produce bacteriocins (ribosomally synthesized antimicrobial peptides) and bile salt hydrolase (BSH), besides being able to survive under gastrointestinal conditions such as high acidity, presence of bile salts, and digestive enzymes. These traits aid enterococci to reach and colonize the intestine, where they may exert beneficial effects. \u003cem\u003eEnterococcus\u003c/em\u003e sp. also attracts the attention of the food industry as a biopreservative culture [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite these promising characteristics, \u003cem\u003eE. faecium\u003c/em\u003e is not widely used in food and feed in part due to its association with nosocomial infections and multidrug resistance[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the gene transfer capability of \u003cem\u003eEnterococcus\u003c/em\u003e species is of special concern.\u003c/p\u003e\u003cp\u003eThe significance of \u003cem\u003eEnterococcus\u003c/em\u003e sp. in foods may be related to its role as an indicator of inadequate sanitary conditions. Nevertheless, previous studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] affirmed that enterococci that lack virulence factors, can be considered safe for use as probiotics. Therefore, the use of \u003cem\u003eE. faecium\u003c/em\u003e for human consumption requires the evaluation of pathogenicity aspects of each strain, according to FAO and WHO [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. From this perspective, the present study aimed to identify and to evaluate, through \u003cem\u003ein vitro\u003c/em\u003e assays, the probiotic properties of \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, isolated from a sample of semi-hard cheese commercialized in Barreiras, Bahia, Brazil. The probiotic candidate strain was also microencapsulated by spray-drying and evaluated with regard to its technological potential.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Bacterial strains\u003c/h2\u003e\u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e EM03 was isolated from semi-hard cheese. The cheese samples were homogenized in a 0.9% (w/v) NaCl solution, tenfold diluted, and plated onto MRS agar. Following incubation at 37\u0026deg;C under anaerobic conditions, typical LAB colonies were selected and subsequently inoculated into MRS broth. One of the isolates was identified as a Gram-positive, catalase-negative, cocci-shaped lactic acid bacterium. It was chosen for further characterization due to its antilisterial activity. Other strains used in this study are listed on Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All the strains were maintained at -40\u0026deg;C in BHI or MRS broth (Kasvi, Brazil) containing 20% (v/v) glycerol (Synth, Brazil).\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\u003eStrains used during this study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCulture Media\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eIncubation temperature\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e NCTC 11954\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e NCTC 12967\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThis study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e NTC 13368\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eListeria monocytogenes\u003c/em\u003e NCTC 13627\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePediococcus acidilactici\u003c/em\u003e LK08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOur collection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMRS\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e NCTC 12903\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e Enteritidis NCTC 6679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Culture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e NCTC 12493\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNational Collection of Type Cultures\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBHI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026deg;C\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e \u003cem\u003eNational Collection of Type Cultures\u003c/em\u003e, United Kingdom.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e BHI broth (\u003cem\u003eBrain Heart Infusion\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e MRS broth (De Man, Rogosa, and Sharpe).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Genotypic identification\u003c/h2\u003e\u003cp\u003eGenotypic identification was performed by sequencing the \u003cem\u003e16S rRNA\u003c/em\u003e gene and for this purpose, genomic DNA was extracted using the kit Wizard DNA Genomic Purification (Promega, Madison, WI, USA). Next, the samples were amplified and purified using the kit Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA). The DNA extracted was used as a template for identification based on the polymerase chain reaction (PCR) carried out in a third-party laboratory, using the primer forward 27F [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] from Thermo Fisher Scientific, S\u0026atilde;o Paulo, Brazil. Purified PCR products were sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems, USA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were analyzed using Chromas Lite 2.1 (Technelysium, South Brisbane, Australia) and compared with sequences available in GenBank using the BLASTN search program from the National Center for Biotechnology Information (\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).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Genotypic characterization\u003c/h2\u003e\u003cp\u003eTotal genomic DNA of \u003cem\u003eE. faecium\u003c/em\u003e EM03 was extracted from a 24 h BHI culture heated at 55\u0026deg;C for 2 h with agitation. Cells were collected by centrifuging 1.5 mL of growth medium at 10,000 x\u003cem\u003eg\u003c/em\u003e for 1 min, then resuspended in 200 \u0026micro;L of ALR-PK buffer with 4 \u0026micro;L of lysozyme. After 90 min of incubation at 56\u0026deg;C, DNA was extracted using the SAMPLE FLEX phT kit (Phoneutria NA2110, Sigma-Aldrich, Missouri, USA) as per the manufacturer\u0026rsquo;s instructions. Extracted DNA quality was checked by spectrophotometry with a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Massachusetts). Samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C for later use.\u003c/p\u003e\u003cp\u003eGene prevalence was determined by PCR for each gene group described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The amplification reaction included 1 \u0026micro;L of genomic DNA, 2.0 \u0026micro;L of 10X buffer, 0.2 mM of each dNTP (Cellco, Brazil), 1 U Taq DNA polymerase (Cellco), and 1 \u0026micro;L of a primer pair mixture in a 20 \u0026micro;L final volume. Amplification began with 5 min at 94\u0026deg;C, followed by 30 cycles of 60 s at 95\u0026deg;C, 60 s at the group-specified annealing temperature (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and 60 s at 72\u0026deg;C. The reaction concluded with a final extension of 10 min at 72\u0026deg;C. Amplicons were analyzed by electrophoresis on a 2% agarose gel containing 1 \u0026micro;L of ethidium bromide. Images were captured using an Lpix Image photodocumentation system (Loccus Biotecnologia, Brazil).\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\u003eOligonucleotide sequences of the primers used in this study\u003csup\u003ea\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget genes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOligonucleotide sequences (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAnnealing temperature (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAmplicon\u003c/p\u003e\u003cp\u003e(pb)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eVirulence genes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehyl\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: ACA GAA GAC CTG CAG GAA ATG\u003c/p\u003e\u003cp\u003eRv: GAC TGA CGT CCA AGT TTC CAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eace\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GAA TTG AGC AAA AGT TCA ATC G\u003c/p\u003e\u003cp\u003eRv: GTC TGT CTT TTC ACT TGT TTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003easa\u003c/em\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GCA CGC TAT TAC GAA CTA TGA\u003c/p\u003e\u003cp\u003eRv: TAA GAA AGA ACA TCA CCA CGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecyl\u003c/em\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: ACT CGG GGA TTG ATA GGC\u003c/p\u003e\u003cp\u003eRv: GCT GCT AAA GCT GCG CTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eefa\u003c/em\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GAC AGA CCC TCA CGA ATA\u003c/p\u003e\u003cp\u003eRv: AGT TCA TCA TGC TGT AGT A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003en.d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eesp\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: AGA TTT CAT CTT TGA TTC TTG G\u003c/p\u003e\u003cp\u003eRv: AAT TGA TTC TTT AGC ATC TGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e510\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003egel\u003c/em\u003eE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: TAT GAC AAT GCT TTT TGG GAT\u003c/p\u003e\u003cp\u003eRv: AGA TGC ACC CGA AAT AAT ATA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e213\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBiogenic amines genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ehdc1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: AGA TGG TAT TGT TTC TTA TG\u003c/p\u003e\u003cp\u003eRv: AGA CCA TAC ACC ATA ACC TT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e367\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003etdc\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GAY ATN ATN GGN ATN GGN YTN GAY CAR G\u003c/p\u003e\u003cp\u003eRv: CCR TAR TCN GGN ATA GCR AAR TCN GTR TG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eodc\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GTN TTY AAY GCN GAY AAR CAN TAY TTY GT\u003c/p\u003e\u003cp\u003eRv: ATN GAR TTN AGT TCR CAY TTY TCN GG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1446\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBacteriocin genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003epedA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: CAA GAT CGT TAA CCA GTT T\u003c/p\u003e\u003cp\u003eRv: CCG TTG TTC CCA TAG TCT AA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003elanM\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: ATG CWA GWY WTG CWC ATG G\u003c/p\u003e\u003cp\u003eRv: CCT AAT GAA CCR TRR YAY CA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u0026ndash;300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003elanB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: TAT GAT CGA GAA RYA KAW AGA TAT GG\u003c/p\u003e\u003cp\u003eRv: TTA TTA IRC AIA TGI AYD AWA CT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e400\u0026ndash;500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003elanC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: TAA TTT AGG ATW ISY IMA YGG\u003c/p\u003e\u003cp\u003eRv: ACC WGK III ICC RTR RCA CCA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u0026ndash;300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eentA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: CAT CAT CCA TAA CTA TAT TTG\u003c/p\u003e\u003cp\u003eRv: AAA TAT TAT GGA AAT GGA GTG TAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e126\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eentB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GAA AAT GAT CAC AGA ATG CCT A\u003c/p\u003e\u003cp\u003eRv: GTT GCA TTT AGA GTA TAC ATT TG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eentP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: TAT GGT AAT GGT GTT TAT TGT AAT\u003c/p\u003e\u003cp\u003eRv: ATG TCC CAT ACC TGC CAA AC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eL50AB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: STG GGA GCA ATC GCA AAA TTA G\u003c/p\u003e\u003cp\u003eRv: ATT GCC CAT CCT TCT CCA AT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAS48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFw: GAG GAG TIT CAT GAT TTA AAG A\u003c/p\u003e\u003cp\u003eRv: CAT ATT GTT AAA TTA CCA AGC AA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSpecies-specif genes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEfaecium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTAGAGACATTGAATATGCC\u003c/p\u003e\u003cp\u003eTCGAATGTGCTACAATC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eY = C or T; R\u0026thinsp;=\u0026thinsp;A or G; N\u0026thinsp;=\u0026thinsp;A, C, G or T.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Antibiotic susceptibility test\u003c/h2\u003e\u003cp\u003eThe antibiotic susceptibility of \u003cem\u003eE. faecium\u003c/em\u003e EM03 was assessed using the disc diffusion method on MRS agar plates that were inoculated with of approximately 10\u003csup\u003e5\u003c/sup\u003e CFU/mL from an overnight culture of \u003cem\u003eE. faecium\u003c/em\u003e EM03 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Antibiotic discs (Laborclin, Pinhais, Brazil) were placed on the surface of the inoculated MRS agar, and the plates were incubated at 37\u0026deg;C for 24 h. For this analysis, it was used antibiotic discs containing amoxicillin (10 \u0026micro;g), ampicillin (10 \u0026micro;g), cefalotin (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), ciprofloxacin (5 \u0026micro;g), clindamycin (2 \u0026micro;g), erythromycin (15 \u0026micro;g), gentamicin (10 \u0026micro;g), tetracycline (30 \u0026micro;g), and vancomycin (30 \u0026micro;g). According to the resistance criteria established by Charteris et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the strain was classified as resistant if the inhibition zone was less than 18 mm for amoxicillin, 12 mm for ampicillin, 14 mm for cefalotin, 13 mm for chloramphenicol, 13 mm for ciprofloxacin, 8 mm for clindamycin, 13 mm for erythromycin, 12 mm for gentamicin, 14 mm for tetracycline, and 14 mm for vancomycin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Evaluation of the probiotic potential\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1 Bacteriocin production and antimicrobial spectrum\u003c/h2\u003e\u003cp\u003eThe antilisterial activity of \u003cem\u003eE. faecium\u003c/em\u003e EM03 was assessed using the spot-on-the-lawn method, as described by Lewus et al [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A volume of 2 \u0026micro;L from a 24 h culture grown in MRS Broth (Kasvi, Brazil) was deposited over TSA-ye (supplemented with 0.6% w/v yeast extract) agar plates (Kasvi, Brazil). After incubating the plates at 37 \u0026ordm;C for 48 h under anaerobic conditions (Probac, Brazil), they were overlaid with 7 mL of molten soft BHI agar (0.8% w/v agar) inoculated with approximately 10\u003csup\u003e6\u003c/sup\u003e CFU/mL of bacterial strains listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Plates were further incubated for 24 h at 37 \u0026ordm;C, and the presence of inhibition halos indicated the production of antimicrobial compounds.\u003c/p\u003e\u003cp\u003eTo verify whether the antimicrobial compound produced by \u003cem\u003eE. faecium\u003c/em\u003e EM03 was a bacteriocin, its susceptibility to proteolytic enzyme degradation was evaluated [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A volume of 2 \u0026micro;L from a 24 h culture grown in MRS Broth (Kasvi, Brazil) was deposited over TSA-ye (supplemented with 0.6% w/v yeast extract) agar plates (Kasvi, Brazil). After incubating the plates at 37 \u0026ordm;C for 48 h under anaerobic conditions (Probac, Brazil), 10 \u0026micro;L of a 20 mg/mL sterile solution of α-Chymotrypsin from bovine pancreas (Sigma-Aldrich, USA) was applied in 2 mm diameter wells on the agar, positioned near the inhibition halo. Water was used as a negative control. The plates were then incubated for 2 h at 25 \u0026ordm;C and, afterward, they were overlaid with 7 mL of molten soft BHI agar (0.8% w/v agar) inoculated with \u003cem\u003eListeria monocytogenes\u003c/em\u003e NCTC 136270 (approximately 10\u003csup\u003e6\u003c/sup\u003e CFU/mL).\u003c/p\u003e\u003cp\u003eAfter 24 h of incubation at 37 \u0026ordm;C, the absence of an inhibition halo around the well containing proteolytic enzyme indicated the proteinaceous nature of the antimicrobial compound (\u003cem\u003ei.e.\u003c/em\u003e bacteriocin).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2 Resistance to low pH and bile salts\u003c/h2\u003e\u003cp\u003eThe survival of \u003cem\u003eE. faecium\u003c/em\u003e EM03 in harsh conditions was evaluated as described by Tulini et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with modifications, as this tolerance represents a critical property for probiotic potential. For this assay, 4 mL of a 24 h BHI broth culture was centrifuged at 2,800 xg for 10 minutes. After removing the supernatant, the pellets were washed with 0.9% w/v NaCl solution and resuspended in 4 mL of the following test media: (i) BHI broth adjusted to pH 2.0, 2.5, or 3.5 with acid; (ii) BHI broth containing 0.3% w/v bile salts (Sigma-Aldrich, USA); (iii) 0.9% w/v NaCl solution containing 3 g/L pepsin and adjusted to pH 2.0; (iv) unmodified BHI broth as a control. The test tubes were incubated at 37\u0026deg;C, with samples collected at 0, 90, and 180 minutes. For bacterial enumeration, 100 \u0026micro;L from each sample was serially diluted in 900 \u0026micro;L of 0.9% w/v NaCl solution, and 10 \u0026micro;L from each dilution was plated on BHI agar and incubated at 35\u0026deg;C for 48 h [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The test was performed in triplicate, and survival rates were determined based on colony counts (log CFU/mL).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Production and characterization of microparticles loaded with \u003cem\u003eE. faecium\u003c/em\u003e EM03\u003c/h2\u003e\u003cp\u003eInitially, 20 g of Arabic gum and 5 g of inulin were dissolved in 100 mL of distilled water and homogenized using an Ultraturrax (IKA, Staufen, Germany) at 10,000 rpm for 5 minutes. Cell biomass was then harvested from 100 mL of a 24 h BHI broth culture, washed twice with 0.9% w/v NaCl solution, and incorporated into the gum Arabic and inulin solution. The resulting mixture was homogenized at 10,000 rpm for 1 minute using Ultraturrax. Subsequently, the mixture was spray dried using a mini spray dryer (Haurok, Florida, USA) set to an inlet temperature of 140\u0026deg;C, an outlet temperature of 85\u0026deg;C, a feed rate of 10 mL/min (controlled by peristaltic pump), and an airflow rate of 10 L/min, using a 0.7 mm nozzle. The dried product was collected and subjected to characterization analyses. The final yield was assessed by comparing the mass of microparticles collected at the end of the process to the mass of solids incorporated into the formulation. In contrast, the encapsulation efficiency was determined by comparing the total log CFU obtained at the end of the process to the total log CFU present in the formulation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Moisture content and water activity\u003c/h2\u003e\u003cp\u003eThe moisture content of the powders was determined using a moisture analyzer (BEL Engineering, Piracicaba, Brazil) equipped with infrared radiation. Water activity was measured using a Tecnal Lab Master instrument (Piracicaba, Brazil).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Morphology\u003c/h2\u003e\u003cp\u003eThe morphology of microparticles and carrier was evaluated by scanning electronic microscopy (Tescan Vega 4 LMU, Brno, Czech Republic). The microparticles were placed over pieces of double-faced carbon tape, fixed on aluminum stubs and covered with gold for 2 min. Thereafter, images were acquired using 2 keV and current of 50 pA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 Fourier transform infrared spectroscopy (FT-IR)\u003c/h2\u003e\u003cp\u003eThe powder was evaluated by Fourier transform infrared (FT-IR) spectroscopy in the 4,000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e regions, using a Shimadzu IRAffinity-1S FTIR spectrometer (Kyoto, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Particle size\u003c/h2\u003e\u003cp\u003eParticle size distribution of spray-dried powder was determined by laser diffraction using a Mastersizer 2000 (Malvern Instruments, UK) device. Samples were dispersed in ethanol under gentle stirring and diluted until reaching an obscuration of \u003cem\u003eca.\u003c/em\u003e 10%, using the Fraunhofer optical model. All measurements were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.6.5 Resistance to simulated gastrointestinal conditions\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe resistance of microencapsulated and free \u003cem\u003eE. faecium\u003c/em\u003e EM03 cells was assessed using the static INFOGEST in vitro digestion protocol [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], with modifications. Simulated gastric fluid (SGF) was prepared by dissolving 3 g/L pepsin from porcine gastric mucosa (P7000, Sigma-Aldrich, USA) and 5 g/L sodium chloride in sterile distilled water, and the pH was adjusted to 3.0. Simulated intestinal fluid (SIF) was prepared by dissolving 6.4 g/L sodium bicarbonate, 1.28 g/L sodium chloride, 0.239 g/L potassium chloride, 3 g/L bovine bile salts (B3883, Sigma-Aldrich, USA), and 1 g/L pancreatin from porcine pancreas (P7545, Sigma-Aldrich, USA) in sterile distilled water, with the pH adjusted to 7.0.\u003c/p\u003e\u003cp\u003eFor microparticle evaluation, 0.1 g of particles was used. As a control, approximately 10⁹ CFU of double-washed \u003cem\u003eE. faecium EM03\u003c/em\u003e cells were included. During the gastric phase, both free cells and microparticles were resuspended in 10 mL SGF and incubated at 37\u0026deg;C. Aliquots of 250 \u0026micro;L were collected at 0, 45, and 90 minutes. Following the gastric phase, samples were centrifuged, the supernatant was removed, and the pellet was resuspended in 10 mL SIF and homogenized. The mixture was incubated at 37\u0026deg;C for the intestinal phase, and aliquots of 250 \u0026micro;L were collected at 0, 60, and 120 minutes. For bacterial enumeration, 100 \u0026micro;L of each sample was serially diluted in 900 \u0026micro;L of 0.9% (w/v) NaCl solution. Subsequently, 10 \u0026micro;L of each dilution was plated on BHI agar and incubated at 35\u0026deg;C for 48 hours [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. All experiments were performed in triplicate. Cell survival was reported as log CFU based on colony counts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.6.6 Effect of storage temperature\u003c/h2\u003e\u003cp\u003eMicroparticles were stored at 4\u0026deg;C and 25\u0026deg;C for up to 60 days. Samples were collected at 0, 15, 30, 45, and 60 days for bacterial enumeration, as previously described. Water activity, SEM and FT-IR analyses were conducted after 60 days of storage. All experiments were conducted in triplicate, and bacterial survival was expressed as log CFU/g.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, USA). Differences between groups were evaluated using Student\u0026rsquo;s t-test for pairwise comparisons and one-way ANOVA to analyze overall group variations, with a significance level set at 5%. Tukey\u0026rsquo;s post-hoc test was applied following ANOVA to determine specific differences between groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Genotypic identification and characterization\u003c/h2\u003e\n \u003cp\u003eThe strain analyzed in this study was identified as a Gram-positive, coccoid-shaped, catalase-negative microorganism. Genotypic identification was performed through sequencing of the 16S rRNA gene, leading to its classification as \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, and confirmed by the detection of the species-specific gene described in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eIn this study, no virulence genes or genes associated with biogenic amine production were detected in \u003cem\u003eE. faecium\u003c/em\u003e EM03, with the exception of the \u003cem\u003ehyl\u003c/em\u003e gene. This gene encodes hyaluronidase, an enzyme responsible for degrading mucopolysaccharides in connective tissues, which can facilitate the spread of microorganisms or toxins within the host [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Pathogenicity in \u003cem\u003eEnterococcus\u003c/em\u003e strains is usually linked to the presence of multiple virulence genes, along with specific phenotypic traits. The occurrence of these genes can vary depending on the strain being evaluated. Fuka et al. [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e] reported a higher prevalence of virulence genes in \u003cem\u003eE. faecalis\u003c/em\u003e strains compared to \u003cem\u003eE. faecium\u003c/em\u003e, with genes such as \u003cem\u003ecyl\u003c/em\u003eA, \u003cem\u003eefa\u003c/em\u003eA, and \u003cem\u003eesp\u003c/em\u003e being more common in \u003cem\u003eE. faecalis\u003c/em\u003e. In contrast, Tsanasidou et al. [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e] found no presence of the genes \u003cem\u003eace\u003c/em\u003e, \u003cem\u003eesp\u003c/em\u003eA, \u003cem\u003ehyl\u003c/em\u003e, or \u003cem\u003egel\u003c/em\u003eE in any of the nine \u003cem\u003eE. faecium\u003c/em\u003e isolates obtained from food sources. Meanwhile, Kiruthiga et al. [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] identified the \u003cem\u003ehyl\u003c/em\u003e gene exclusively in \u003cem\u003eE. faecium\u003c/em\u003e isolates. This variability in genotypic virulence profiles may be attributed to the ability of bacterial strains to undergo horizontal gene transfer. Based on the results of this study and considering the relatively common occurrence of the \u003cem\u003ehyl\u003c/em\u003e gene in non-pathogenic \u003cem\u003eEnterococcus\u003c/em\u003e strains, it is possible to infer that \u003cem\u003eE. faecium\u003c/em\u003e EM03 does not present any pathogenic traits. Therefore, given its safe phenotypic profile and the genotypic characteristics observed in other cheese-derived strains, \u003cem\u003eE. faecium\u003c/em\u003e EM03 can be regarded as safe for biotechnological applications.\u003c/p\u003e\n \u003cp\u003eInvestigations into the genes responsible for encoding bacteriocins in \u003cem\u003eE. faecium\u003c/em\u003e EM03 revealed the presence of the genes \u003cem\u003eent\u003c/em\u003eB, \u003cem\u003eent\u003c/em\u003eP, L50AB, \u003cem\u003elan\u003c/em\u003eM and \u003cem\u003elan\u003c/em\u003eC. The enterocins produced by \u003cem\u003eEnterococcus\u003c/em\u003e fall under class II bacteriocins and are noted for their strong antimicrobial activities against foodborne bacteria [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Similar findings were reported by Favaro et al. [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e], who detected a combination of enterocin genes in all analyzed \u003cem\u003eE. faecium\u003c/em\u003e strains, including \u003cem\u003eent\u003c/em\u003eA, \u003cem\u003eent\u003c/em\u003eE, \u003cem\u003eent\u003c/em\u003eP, and L50. Additionally, Valledor et al. [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] explored enterocin genes in \u003cem\u003eE. faecium\u003c/em\u003e ST20Kc and ST41Kc, identifying the presence of \u003cem\u003eent\u003c/em\u003eA, \u003cem\u003eent\u003c/em\u003eB, and \u003cem\u003eent\u003c/em\u003eP, which exhibited significant inhibitory effects against \u003cem\u003eL. monocytogenes\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e. Other studies have similarly documented the co-occurrence of enterocin-encoding genes in \u003cem\u003eE. faecium\u003c/em\u003e strains [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. The \u003cem\u003elan\u003c/em\u003eM and \u003cem\u003elan\u003c/em\u003eC genes are implicated in the biosynthesis of lantibiotics, which are peptides that undergo post-translational modifications to attain biological activity, primarily targeting the bacterial cell wall [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Research on these genes is relatively rare among \u003cem\u003eEnterococcus\u003c/em\u003e species, despite the prevalence of lantibiotic production in lactic acid bacteria from the genera \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, and \u003cem\u003eEnterococcus\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although multiple enterocin genes have been confirmed, there is currently no evidence to suggest that all these genes are expressed simultaneously in the examined strain. The detection of several genes may account for the high antimicrobial activity observed against \u003cem\u003eL. monocytogenes\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e, however, further experiments are necessary to evaluate their actual expression levels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Antibiotic Susceptibility\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e EM03 exhibited varying susceptibility profiles to the antibiotics tested: it was sensitive to ampicillin (10 \u0026micro;g), tetracycline (30 \u0026micro;g), amoxicillin (10 \u0026micro;g), vancomycin (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), and cephalothin (30 \u0026micro;g). The strain showed moderate sensitivity to erythromycin (15 \u0026micro;g) and clindamycin (2 \u0026micro;g), while it was resistant to gentamicin (10 \u0026micro;g) and ciprofloxacin (5 \u0026micro;g). Evaluating antimicrobial susceptibility is one of the minimum criteria for considering a strain as a potential probiotic, as in vitro safety assessments are essential [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Antibiotic sensitivity is favorable because it ensures the effective elimination of the microorganism from the host when necessary. Additionally, probiotic candidates should ideally not exhibit significant antibiotic resistance to reduce the risk of transferring resistance genes to the intestinal microbiota. The results indicated high sensitivity of \u003cem\u003eE. faecium\u003c/em\u003e EM03 to critically important antibiotics, such as vancomycin and ampicillin, suggesting a low health risk compared to clinical isolates from hospitalized patients. Similar findings were reported by \u0026Ccedil;etin et al. [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e] for \u003cem\u003eE. faecium\u003c/em\u003e strains isolated from raw cow\u0026rsquo;s milk, which also showed sensitivity to ampicillin, vancomycin, clindamycin, tetracycline, and chloramphenicol, resistance to gentamicin, and intermediate susceptibility to erythromycin. Although antibiotic resistance genes were not investigated in this study, the gentamicin resistance observed in \u003cem\u003eE. faecium\u003c/em\u003e EM03 may represent an intrinsic characteristic of the species, as seen in other studies [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e\n \u003cp\u003eResistance to glycopeptides, particularly vancomycin and ampicillin, is a significant concern when considering the use of \u003cem\u003eEnterococcus\u003c/em\u003e spp. in food applications. This resistance is typically attributed to the presence of peptidoglycan precursors that enhance cell wall rigidity, thereby hindering the effectiveness of these antibiotics [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, \u003cem\u003eE. faecium\u003c/em\u003e EM03 demonstrated a high sensitivity to these glycopeptide antibiotics, indicating the absence of genes associated with peptidoglycan modification, and thereby supporting the efficacy of a broad array of antimicrobials. Corroborating this susceptibility phenotype, Cariolato et al. [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e] reported that \u003cem\u003eE. faecium\u003c/em\u003e strains isolated from food sources exhibited greater susceptibility to glycopeptides compared to clinical isolates. Therefore, the differences in resistance patterns observed among \u003cem\u003eEnterococcus\u003c/em\u003e spp. strains may be linked to their source of isolation. Clinical strains frequently display multidrug resistance due to the acquisition of resistance genes through conjugation, which is often facilitated by interactions with pathogenic bacteria colonizing the intestinal tracts of hospitalized patients.\u003c/p\u003e\n \u003cp\u003eResistance to ciprofloxacin observed in some bacterial strains may result from mutations in genes that encode target enzymes affected by the antibiotic, thus reducing its affinity. This association has been documented in \u003cem\u003eEnterococcus\u003c/em\u003e species [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Nevertheless, resistance to fluoroquinolones is typically anticipated in \u003cem\u003eE. faecium\u003c/em\u003e, primarily due to the production of the chromosomal enzyme aac(6\u0026rsquo;)-I, which diminishes the effectiveness of aminoglycosides [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIn conclusion, \u003cem\u003eE. faecium\u003c/em\u003e EM03 demonstrates an antimicrobial susceptibility pattern typical of non-pathogenic strains within the species, displaying no resistance to important antibiotics used in clinical settings and indicating its safety for use as a probiotic.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Evaluation of the probiotic potential\u003c/h2\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1 Bacteriocin production and antimicrobial spectrum\u003c/h2\u003e\n \u003cp\u003eThe assay performed with \u003cem\u003eE. faecium\u003c/em\u003e EM03 exhibited a significant inhibitory effect against \u003cem\u003eListeria monocytogenes\u003c/em\u003e NCTC 136270, producing an average inhibition zone of 22.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 mm in diameter. Additionally, it demonstrated activity against \u003cem\u003eEnterococcus faecali\u003c/em\u003es NCTC 12967, resulting in a halo measuring 14.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm. However, no inhibitory effect was observed against the other tested indicators. Moreover, it was noted that the antimicrobial compound is of a proteinaceous nature (specifically, a bacteriocin), as indicated by the degradation of the inhibition halo upon treatment with protease (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eEvaluating the antagonistic activity against pathogens is essential in assessing the functional properties of probiotic candidates. The synthesis of bacteriocins plays a crucial role, as it helps curb the proliferation of pathogenic bacteria and supports the stability of the intestinal microbiota. Previous studies, such as those by Charyyev et al.[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e] and Nami et al. [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e], have reported strong inhibition of \u003cem\u003eL. monocytogenes\u003c/em\u003e by various \u003cem\u003eE. faecium\u003c/em\u003e strains, with inhibition zones of approximately 23 mm, which aligns closely with the results of the present study. Additionally, inhibitory activity against \u003cem\u003eE. faecalis\u003c/em\u003e was noted by Furlaneto-Maia et al. [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e], who reported halos of 10 mm or less. Although these values are lower than those observed in this study, they further emphasize the protective potential of \u003cem\u003eE. faecium\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThe results affirm the effectiveness of the bacteriocins (enterocins) produced by \u003cem\u003eE. faecium\u003c/em\u003e EM03 in inhibiting foodborne pathogens. The notable antilisterial activity observed is characteristic of class IIa enterocins, which are also recognized for their robust resistance to high temperatures [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. The mechanisms by which these bacteriocins operate involve altering the permeability of pathogenic bacterial cell membranes, ultimately leading to cell lysis[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this context, the effects of enterocins have been extensively researched, and their efficacy is viewed as a promising alternative for safeguarding foods against bacterial contamination. Zhou et al. [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e] assessed the enterocins produced by \u003cem\u003eE. faecium\u003c/em\u003e B1 in food matrices and reported significant inhibitory activity against \u003cem\u003eL. monocytogenes\u003c/em\u003e, comparable to that of the bacteriocins synthesized by \u003cem\u003eE. faecium\u003c/em\u003e EM03. Moreover, antilisterial activity has also been demonstrated by Schittler et al. [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e] and Popović et al. [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e], who reported positive outcomes from the use of \u003cem\u003eE. faecium\u003c/em\u003e in the production of fermented foods and in the preservation of milk. This efficacy is particularly attributed to its biocontrol capabilities against pathogens and its ability to reduce the viable count of \u003cem\u003eL. monocytogenes\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eAlthough the purification and characterization of the bacteriocins produced by \u003cem\u003eE. faecium\u003c/em\u003e EM03 were not conducted, the synthesis of these substances is corroborated by the detection of enterocin-encoding genes (\u003cem\u003eent\u003c/em\u003eA, \u003cem\u003eent\u003c/em\u003eP, L50A, and L50B). Further evaluations conducted by Mancini et al. [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] and Valledor et al. [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] on \u003cem\u003eE. faecium\u003c/em\u003e species also identified these genes and demonstrated significant antilisterial activity, corroborating the findings of this study. These results underscore the functional potential of \u003cem\u003eE. faecium\u003c/em\u003e EM03 as a viable alternative as a protective agent against food related bacteria. This antimicrobial property is advantageous for the selection of probiotics within the \u003cem\u003eEnterococcus\u003c/em\u003e genus, enabling specific strains to be utilized for pathogen control in the gastrointestinal tract.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2 Resistance to low pH and bile salts\u003c/h2\u003e\n \u003cp\u003eIn the present study, \u003cem\u003eE. faecium\u003c/em\u003e EM03 exhibited a significant decline in viability at pH 2.0 during the first 90 minutes, maintaining partial stability up to 180 minutes, which suggests greater sensitivity to extremely acidic conditions. Resistance at pH 2.5 was notably higher than at pH 2.0, with a stable count of 7.3 log CFU/mL during the initial 90 minutes and only slight variation thereafter, indicating mild sensitivity to the gastric environment during prolonged exposure. The highest survival rate was recorded at pH 3.5, with counts ranging from 7.4 to 8 log CFU/mL throughout the 180 minutes, showing some fluctuation up to 90 minutes before stabilizing. The ability to endure acidic conditions and traverse the gastrointestinal tract is one of the primary challenges for probiotic strains. In this context, the gastric environment serves as the first biological barrier, with pH levels ranging from 2.5 to 3.5, thereby limiting bacterial survival [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eIn the presence of pepsin, the strain demonstrated low resistance during the 90-minute exposure, with counts ranging from 7.3 to 8.5 log CFU/mL. Notably, there was a more significant decline in counts at the end of this period compared to the control group. Conversely, when subjected to 0.3% bile, the count remained stable at approximately 8.5 log CFU/mL over the 180 minutes, showing no significant reductions. This indicates a high viability and adaptation to intestinal conditions (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThese findings are consistent with studies by Gupta et al. [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e], Ankaiah et al. [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e] and Xiao et al. [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e], which also noted low resistance of \u003cem\u003eE. faecium\u003c/em\u003e WFD-128 to extremely acidic environments, indicating that conditions above pH 2.5 are more conducive to survival. In contrast, Bhardwaj et al. [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e] found that some \u003cem\u003eEnterococcus\u003c/em\u003e strains exhibited resistance at pH 2.0, though the highest survival rates were observed under milder conditions, particularly between pH 3.0 and 3.5. While certain studies characterize \u003cem\u003eE. faecium\u003c/em\u003e as resistant to highly acidic environments [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e], the majority indicate significant growth only at pH values exceeding 2.5. This suggests that \u003cem\u003eE. faecium\u003c/em\u003e strains exhibit a high level of acid tolerance, a trait often linked to their ability to maintain pH homeostasis through proton extrusion, thus preserving cell membrane integrity [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. Nevertheless, proliferation diminishes as acidity increases, which helps elucidate the growth performance of specific Enterococcus strains in fermented foods. Consequently, E. faecium EM03 shows a considerable ability to survive gastrointestinal passage. Strategies such as microencapsulation or co-administration with foods that can modulate gastric pH may further enhance its viability and probiotic efficacy.\u003c/p\u003e\n \u003cp\u003eThe resistance of \u003cem\u003eE. faecium\u003c/em\u003e EM03 to pepsin and 0.3% bile enables its effective passage and colonization within the intestinal environment in viable amounts, which is crucial for the expression of probiotic functions. Typically, bile impacts bacterial survival by interacting with surface proteins; however, species from the \u003cem\u003eEnterococcus\u003c/em\u003e genus have developed mechanisms to mitigate this effect [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. Similar findings were reported by Tilwani et al. [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e], who observed that \u003cem\u003eE. faecium\u003c/em\u003e MC-5 maintained its viability, particularly in the presence of 0.3% bile. Likewise, Bhardwaj et al. [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e] noted that \u003cem\u003eE. faecium\u003c/em\u003e KH24 did not exhibit significant variations in viability when exposed to various bile concentrations (1%, 2%, and 3%). The bile salt tolerance observed in this study, along with previous research findings, underscores the potential application of \u003cem\u003eE. faecium\u003c/em\u003e EM03 as a probiotic.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Production and characterization of microparticles loaded with \u003cem\u003eE. faecium\u003c/em\u003e EM03\u003c/h2\u003e\n \u003cp\u003eMicroparticles containing \u003cem\u003eE. faecium\u003c/em\u003e EM03 were produced through a spray-drying process, using Arabic gum and inulin as wall materials. The final yield of the microparticles collected from the cyclone compartment of the spray dryer was 39.5%, with an encapsulation efficiency of 94.0% and a bacterial population of 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 log CFU/g. The resulting powder had a moisture content of 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 g/100g and 0.320\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 of water activity.\u003c/p\u003e\n \u003cp\u003eMoisture content indicates the total amount of water present in microparticles, while water activity refers to the portion of water that is available for microbial growth and chemical reactions. Both parameters are essential for ensuring the stability of probiotics during storage. Ideally, moisture levels should range between 4% and 7% to maintain stability and prolong the product\u0026apos;s shelf life [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. According to Barbosa et al. [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e], lower moisture levels can reduce particle agglomeration and promote better dispersion in food matrices. It\u0026apos;s also important to highlight that inulin is well-known for its ability to retain water, often forming gel-like structures when hydrated [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e]. However, its inclusion in the present formulation did not significantly increase moisture content to a level that would adversely affect the rheology of the particles.\u003c/p\u003e\n \u003cp\u003eThe optimal water activity (A\u003csub\u003ew\u003c/sub\u003e) for probiotics is typically recommended to be below 0.2, as this threshold promotes bacterial survival and mitigates the risk of product contamination during storage [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e]. In this study, the measured A\u003csub\u003ew\u003c/sub\u003e exceeded the ideal value, but is equivalent to the values reported by Ram\u0026iacute;rez-Dami\u0026aacute;n et al. [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e], Reyes et al. [\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e], and Naseem et al. [\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e] who developed encapsulated microparticles using Arabic gum alone or in combination with inulin. Their results revealed water activity similar to those found in this research, ranging from 0.31 to 0.33, which also surpassed the recommended range, probably due to the presence of inulin.\u003c/p\u003e\n \u003cp\u003eThe microparticles displayed a primarily spherical morphology, typical of spray-dried systems, as presented in Fig.\u0026nbsp;3. Their surfaces appear irregular and wrinkled, with several particles exhibiting concavities or collapsed areas, also a common characteristic due to rapid moisture loss during atomization. Additionally, the sample contains numerous smaller satellite particles adhered to the surfaces of larger ones, resulting in an agglomerated structure. Overall, the microstructure indicates effective particle formation, characterized by heterogeneous sizes and typical shrinkage-related depressions observed in polysaccharide-based microcapsules containing inulin and gum Arabic. In 2021, Karrar et al. [\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e] encapsulated gurum seed oil by spray-drying using formulations with Arabic gum and whey protein isolate, and obtained very similar results when observing the microparticles by SEM.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the Fourier transform infrared spectroscopy (FT-IR) analyses revealed distinct absorption peaks, notably around 3,300 cm⁻\u0026sup1;, corresponding to O\u0026ndash;H stretching vibrations. A peak near 1,600 cm⁻\u0026sup1; is attributed to C\u0026thinsp;=\u0026thinsp;O stretching, while another peak around 1,000 cm⁻\u0026sup1; suggests C\u0026ndash;O stretching or C\u0026ndash;O\u0026ndash;C vibrations, which are typically associated with glycosidic bonds. The similarities observed between the spectra of the wall material and the microparticles indicate that there was no chemical interaction between the encapsulated material and the wall material. Similar results were also reported by Hoang et al. [\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e] for the srpay-drying encapsulation of Hibiscus sabdariffa extract using maltodextrin and Arabic gum.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the particle size distribution of microparticles loaded with \u003cem\u003eE. faecium\u003c/em\u003e EM03, averaging 10.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80 \u0026micro;m. The volume-weighted mean diameter (D[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]) was 11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 \u0026micro;m, while the surface area-weighted mean diameter (D[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]) was 5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u0026micro;m, resulting in a span of 1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;m. D[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e] highlights the presence of larger particles in the sample, with higher values indicating a greater contribution from these larger particles. In contrast, D[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e] emphasizes smaller particles, which are particularly important for surface-dependent processes, as lower values correspond to finer particles that possess a larger total surface area. The Span, calculated as (D90 \u0026ndash; D10) / D50, provides insight into the breadth of the particle size distribution; lower values signify a narrower distribution, whereas higher values indicate a broader one. Together, these parameters are crucial for characterizing average particle size and polydispersity [\u003cspan class=\"CitationRef\"\u003e77\u003c/span\u003e]. From a technological perspective, larger particles may enhance probiotic protection during atomization, as bigger droplets tend to heat more slowly and lose water more gradually during spray drying, reducing thermal and dehydration stress on the cells. In this study, the span value (1.61) suggests a moderately broad yet acceptable distribution, which is commonly observed in polysaccharide-based spray-dried systems and is unlikely to impair powder flowability or application. In 2025, Fahrudin et al. [\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e] reported an average particle size of approximately 5.2 \u0026micro;m, with D[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e] measuring 4.45 \u0026micro;m for microparticles produced from Arabic gum and vegetable extracts. The smaller particles reported by Fahrudin et al. [\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e] may also be related to the use of Arabic gum as the sole wall material. In contrast, the present formulation combined 20% Arabic gum with 5% inulin, which increases the feed viscosity and typically leads to the formation of larger droplets during atomization. This effect has been widely described in spray-dried polysaccharide systems, where higher viscosity promotes larger particle sizes and broader distributions. Also, this variation in values may stem from differences in equipment and drying parameters, which directly affect the size and morphology of the resulting powder.\u003c/p\u003e\n \u003cp\u003eThe resistance of both free and encapsulated bacteria to simulated gastrointestinal conditions was assessed, with results illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The population of free bacteria began to decline after 90 minutes, along with the conclusion of the gastric phase. In contrast, the population of microencapsulated bacteria started to decrease after 45 minutes, during the mid-gastric phase. Both types of situations exhibited remarkable survival rates, with 86% surviving by the end of the assay. These findings suggest that the microencapsulation of \u003cem\u003eE. faecium\u003c/em\u003e EM03 could serve as an effective technological approach to enhance bacterial survival throughout a product\u0026apos;s shelf life, thereby facilitating its industrial application, as powders are generally easier to handle.\u003c/p\u003e\n \u003cp\u003eThe evaluations conducted by Paula et al. [\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e] demonstrated that Arabi gum significantly enhanced the survival of \u003cem\u003eL. plantarum\u003c/em\u003e strains, with viability rates reaching 80% for microencapsulated cells compared to their free counterparts. However, a decrease in viability during the gastric phase was noted, attributable to the acidic conditions. Additionally, the favorable outcomes associated with the incorporation of inulin into the encapsulating matrix align with the findings of Ahmadi et al. [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e] and Meral et al. [\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e]. These studies also observed an increased viability of probiotic bacteria, specifically \u003cem\u003eL. acidophilus\u003c/em\u003e LA-5 and \u003cem\u003eL. rhamnosus\u003c/em\u003e, when these strains were encapsulated with inulin, offering superior protection compared to those encapsulated without this compound. Consequently, the combined use of Arabic gum and inulin as wall materials for encapsulation may provide enhanced protection for probiotic strains compared to the use of either material in isolation, as evidenced by this study and other research [\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1 Effect of storage temperature\u003c/h2\u003e\n \u003cp\u003eMicroparticles were stored at temperatures of 4\u0026deg;C and 25\u0026deg;C for up to 60 days, and bacterial counts were performed on samples taken at 0, 15, 30, 45, and 60 days. In addition, analyses of water activity, SEM, FT-IR and size distribution were conducted following the 60-day storage period. The resulting data is presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, and 10.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBacterial population, water activity average size, volume-weighted mean diameter (D[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]), surface area-weighted mean diameter (D[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]) and span of microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin. The microparticles were stored at 4\u0026deg;C and 25\u0026deg;C for up to 60 days. Different letters in a row indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0 days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e15 days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30 days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e45 days\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e60 days\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u0026deg;C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBacterial population (log CFU/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.320\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.345\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage size (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e] (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e] (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpan (\u0026micro;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003e25\u0026deg;C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBacterial population (log CFU/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater activity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.320\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.467\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage size\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003en.d.: not determined\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe data presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e regarding the bacterial population suggests that the formulation stored at 4\u0026deg;C for 60 days successfully maintained its original bacterial count. In contrast, the formulation stored at 25\u0026deg;C for the same duration exhibited a slight decline in bacterial population compared to the initial count. This reduction can likely be attributed to higher temperatures, as 25\u0026deg;C is more favorable for bacterial metabolism than 4\u0026deg;C. Furthermore, it is noteworthy that the water activity (A\u003csub\u003ew\u003c/sub\u003e) at 25\u0026deg;C increased, which may have further enhanced bacterial metabolism during storage. Overall, the stability observed during storage can also be attributed to the presence of prebiotics within the matrix, which contributed to the viability of the bacteria. These findings suggest that the synergistic effect of Arabic gum and inulin offers protection and enhances the durability of probiotics during storage.\u003c/p\u003e\n \u003cp\u003eStudies utilizing inulin as encapsulating agent for \u003cem\u003eL. acidophilus\u003c/em\u003e have reported positive effects on bacterial stability, achieving high viability rates of 7.72 log CFU/g compared to particles lacking inulin [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e]. Similarly, Naseem et al. [\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e] highlighted the effectiveness of Arabic gum as a protective material against environmental factors, resulting in probiotic microparticles exhibiting higher survival rates and thermal stability. Additionally, findings from Yin et al. [\u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e] indicated that encapsulation using Arabic gum remained effective when stored at 4\u0026deg;C for 16 weeks, showing only a reduction of less than 1.5 log CFU/g. However, a decline in viable cells was noted when exposed to 25\u0026deg;C, reflecting patterns similar to those observed in the present study. In conclusion, the data suggests that the combination of Arabic gum and inulin as encapsulating agents provides enhanced protection against external factors, with inulin also supporting the survival of probiotic bacteria due to its prebiotic properties.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e revealed that the morphological structures kept visuable the same for particles stored at 4 and 25\u0026deg;C, and also very similar to the particles presented in Fig. 3 immediately after their production. Figure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e also indicates that there was no change in the chemical interactions between the components of microparticles, neither a change in the spectra pattern. Similarly, Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e indicates the same pattern of size differences for particles store at both conditions, although Table 4 showed an increase on D[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e] and D[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e] values for particles stored at 4\u0026deg;C, which may be attributed to the low temperature of storage. Taken together, these results suggests that microparticles loaded with \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, obtained by spray-drying a formulation composed of 20% Arabic gum and 5% inulin, may be successfully stored at 4\u0026deg;C for up to 60 days without compromising microbiological and physical parameters.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003e\u003cem\u003eE. faecium\u003c/em\u003e EM03 exhibits significant technological potential for protecting against foodborne pathogens, as evidenced by the presence of genes responsible for the production bacteriocins, its strong antilisterial activity, and its probiotic properties, which are demonstrated by its ability to withstand conditions simulating passage through the gastrointestinal tract. The safety assessment indicated no resistance to clinically important antibiotics and absence of genes for the production of virulence factors and biogenic amines. Additionally, the application of the spray-drying encapsulation technique utilizing the Arabic gum\u0026ndash;inulin complex as a wall matrix has proven effective in enhancing the strain's viability and stability during storage, as well as protecting it from adverse conditions and enzymatic action. Collectively, these findings suggest that \u003cem\u003eE. faecium\u003c/em\u003e EM03 possesses broad functional potential that remains largely unexplored, particularly concerning the enterocins it produces and its probiotic effects, including bile salt hydrolase activity. Therefore, future studies are crucial to deepen our understanding of its functionalities, facilitate the discovery of new applications, and expand the scientific knowledge surrounding this species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no competing interests. The data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Dennis Coelho Cruz for assistance with SEM image acquisition and MCTI/FINEP/FNDCT/CT-INFRA facilities for instrumental analyses. The research leading to these results received funding from CNPq (National Council for Scientific and Technological Development: 403613/2023-0), and FAPESB (Foundation for Research Support of the State of Bahia: BioproFar-BA PIE0001/2024; PPF0004/2023; PIE0006/2022; PPF0017/2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.S.O.M. conducted microbiological, genetic, and physical analyses, drafted the manuscript, and contributed to its revision. G.E.N.O., B.P.S. and M.P.S.P. performed microparticle analyses and contributed to manuscript preparation. I.R. executed the genetic sequencing analyses. E.C.P.D.M. supervised the genetic sequencing analyses and contributed to both writing and revising the manuscript. I.A.G. also contributed to the writing and revision of the manuscript. M.H.F.K. carried out and supervised the genotyping analyses, and contributed to the manuscript writing and revision. F.L.T. managed the project, provided funding, and contributed to the writing and revision of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFAO, WHO (2002) Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas TK, Pradhan S, Chakrabarti S et al (2022) Current status of probiotic and related health benefits. Appl Food Res 2:100185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.afres.2022.100185\u003c/span\u003e\u003cspan address=\"10.1016/j.afres.2022.100185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiao W, Chen C, Wen T, Zhao Q (2021) Probiotics for the Prevention of Antibiotic-associated Diarrhea in Adults. J Clin Gastroenterol 55:469\u0026ndash;480. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/MCG.0000000000001464\u003c/span\u003e\u003cspan address=\"10.1097/MCG.0000000000001464\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng J, Wittouck S, Salvetti E et al (2020) A taxonomic note on the genus \u003cem\u003eLactobacillus\u003c/em\u003e: Description of 23 novel genera, emended description of the genus \u003cem\u003eLactobacillus\u003c/em\u003e Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol 70:2782\u0026ndash;2858. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/ijsem.0.004107\u003c/span\u003e\u003cspan address=\"10.1099/ijsem.0.004107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Wang J, Wang M et al (2023) \u003cem\u003eBifidobacterium\u003c/em\u003e: a probiotic for the prevention and treatment of depression. Front Microbiol 14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1174800\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1174800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMulaw G, Tessema TS, Muleta D, Tesfaye A (2019) \u003cem\u003eIn Vitro\u003c/em\u003e Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from Some Traditionally Fermented Ethiopian Food Products. Int J Microbiol 2019:1\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2019/7179514\u003c/span\u003e\u003cspan address=\"10.1155/2019/7179514\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGiraffa G (2002) Enterococci from foods. FEMS Microbiol Rev 26:163\u0026ndash;171. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0168-6445(02)00094-3\u003c/span\u003e\u003cspan address=\"10.1016/S0168-6445(02)00094-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranz CMAP, Van Belkum MJ, Holzapfel WH et al (2007) Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31:293\u0026ndash;310. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6976.2007.00064.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6976.2007.00064.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRotta IS, Rodrigues WF, Santos CTB et al (2022) Clinical isolates of \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eE. faecium\u003c/em\u003e harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants. Microb Pathog 171:105715. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2022.105715\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2022.105715\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGundog DA, Onmaz NE, Gungor C et al (2025) \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eE. faecium\u003c/em\u003e in dairy production line: Antibiotic resistance profile and virulence characteristics. Int Dairy J 165:106209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.idairyj.2025.106209\u003c/span\u003e\u003cspan address=\"10.1016/j.idairyj.2025.106209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes BC, Esteves CT, Palazzo ICV et al (2008) Prevalence and characterization of \u003cem\u003eEnterococcus\u003c/em\u003e spp. isolated from Brazilian foods. Food Microbiol 25:668\u0026ndash;675. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fm.2008.03.008\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2008.03.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurner S, Pryer KM, Miao VPW, Palmer JD (1999) Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis1. J Eukaryot Microbiol 46:327\u0026ndash;338. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1550-7408.1999.tb04612.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1550-7408.1999.tb04612.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115\u0026ndash;175\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVankerckhoven V, Van Autgaerden T, Vael C et al (2004) Development of a Multiplex PCR for the Detection of \u003cem\u003easa1\u003c/em\u003e, \u003cem\u003egelE\u003c/em\u003e, \u003cem\u003ecylA\u003c/em\u003e, \u003cem\u003eesp\u003c/em\u003e, and \u003cem\u003ehyl\u003c/em\u003e Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of \u003cem\u003eEnterococcus faecium\u003c/em\u003e. J Clin Microbiol 42:4473\u0026ndash;4479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/JCM.42.10.4473-4479.2004\u003c/span\u003e\u003cspan address=\"10.1128/JCM.42.10.4473-4479.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraes PM, Perin LM, Todorov SD et al (2012) Bacteriocinogenic and virulence potential of \u003cem\u003eEnterococcus\u003c/em\u003e isolates obtained from raw milk and cheese. J Appl Microbiol 113:318\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2672.2012.05341.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2672.2012.05341.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTulini FL, B\u0026iacute;scola V, Choiset Y et al (2015) Evaluation of the proteolytic activity of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e FT132 and \u003cem\u003eLactobacillus paracasei\u003c/em\u003e FT700, isolated from dairy products in Brazil, using milk proteins as substrates. Eur Food Res Technol 241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00217-015-2470-6\u003c/span\u003e\u003cspan address=\"10.1007/s00217-015-2470-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOmar N, Ben, Castro A, Lucas R et al (2004) Functional and Safety Aspects of Enterococci Isolated from Different Spanish Foods. Syst Appl Microbiol 27:118\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1078/0723-2020-00248\u003c/span\u003e\u003cspan address=\"10.1078/0723-2020-00248\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEaton TJ, Gasson MJ (2001) Molecular Screening of \u003cem\u003eEnterococcus\u003c/em\u003e Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates. Appl Environ Microbiol 67:1628\u0026ndash;1635. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.67.4.1628-1635.2001\u003c/span\u003e\u003cspan address=\"10.1128/AEM.67.4.1628-1635.2001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRivas P, Alonso J, Moya J et al (2005) The Impact of Hospital-Acquired Infections on the Microbial Etiology and Prognosis of Late-Onset Prosthetic Valve Endocarditis. Chest 128:764\u0026ndash;771. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1378/chest.128.2.764\u003c/span\u003e\u003cspan address=\"10.1378/chest.128.2.764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlbano H, Todorov SD, van Reenen CA et al (2007) Characterization of two bacteriocins produced by \u003cem\u003ePediococcus acidilactici\u003c/em\u003e isolated from Alheira, a fermented sausage traditionally produced in Portugal. Int J Food Microbiol 116:239\u0026ndash;247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2007.01.011\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2007.01.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHyink O, Balakrishnan M, Tagg JR (2005) \u003cem\u003eStreptococcus rattus\u003c/em\u003e strain BHT produces both a class I two-component lantibiotic and a class II bacteriocin. FEMS Microbiol Lett 252:235\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.femsle.2005.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.femsle.2005.09.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWirawan RE, Klesse NA, Jack RW, Tagg JR (2006) Molecular and genetic characterization of a novel nisin variant produced by \u003cem\u003eStreptococcus uberis\u003c/em\u003e. Appl Environ Microbiol 72:1148\u0026ndash;1156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.72.2.1148-1156.2006\u003c/span\u003e\u003cspan address=\"10.1128/AEM.72.2.1148-1156.2006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDu Toit M, Franz CMAP, Dicks LMT, Holzapfel WH (2000) Preliminary characterization of bacteriocins produced by \u003cem\u003eEnterococcus faecium\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e isolated from pig faeces. J Appl Microbiol 88:482\u0026ndash;494. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-2672.2000.00986.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-2672.2000.00986.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDutka-Malen S, Evers S, Courvalin P (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jcm.33.1.24-27.1995\u003c/span\u003e\u003cspan address=\"10.1128/jcm.33.1.24-27.1995\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang C-Y, Lin P-R, Ng C-C, Shyu Y-T (2010) Probiotic properties of \u003cem\u003eLactobacillus\u003c/em\u003e strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage. Anaerobe 16:578\u0026ndash;585. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anaerobe.2010.10.003\u003c/span\u003e\u003cspan address=\"10.1016/j.anaerobe.2010.10.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCharteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially probiotic \u003cem\u003eLactobacillus\u003c/em\u003e species. J Food Prot 61:1636\u0026ndash;1643. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4315/0362-028X-61.12.1636\u003c/span\u003e\u003cspan address=\"10.4315/0362-028X-61.12.1636\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLewus CB, Kaiser A, Montville TJ (1991) Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Appl Environ Microbiol 57:1683\u0026ndash;1688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.57.6.1683-1688.1991\u003c/span\u003e\u003cspan address=\"10.1128/aem.57.6.1683-1688.1991\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTulini FL, Winkelstr\u0026ouml;ter LK, De Martinis ECP (2013) Identification and evaluation of the probiotic potential of \u003cem\u003eLactobacillus paraplantarum\u003c/em\u003e FT259, a bacteriocinogenic strain isolated from Brazilian semi-hard artisanal cheese. Anaerobe 22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anaerobe.2013.06.006\u003c/span\u003e\u003cspan address=\"10.1016/j.anaerobe.2013.06.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoben HJ, Somasegaran P (1982) Comparison of the pour, spread, and drop plate methods for enumeration of \u003cem\u003eRhizobium\u003c/em\u003e spp. in inoculants made from presterilized peat. Appl Environ Microbiol 44:1246\u0026ndash;1247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.44.5.1246-1247.1982\u003c/span\u003e\u003cspan address=\"10.1128/aem.44.5.1246-1247.1982\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrodkorb A, Egger L, Alminger M et al (2019) INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat Protoc 14:991\u0026ndash;1014. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41596-018-0119-1\u003c/span\u003e\u003cspan address=\"10.1038/s41596-018-0119-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim Y, Choi S-I, Jeong Y, Kang C-H (2022) Evaluation of safety and probiotic potential of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e MG5206 and \u003cem\u003eEnterococcus faecium\u003c/em\u003e MG5232 isolated from Kimchi, a Korean fermented cabbage. Microorganisms 10:2070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms10102070\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms10102070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChajęcka-Wierzchowska W, Zadernowska A, Łaniewska-Trokenheim Ł (2017) Virulence factors of \u003cem\u003eEnterococcus\u003c/em\u003e spp. presented in food. LWT 75:670\u0026ndash;676. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2016.10.026\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2016.10.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuka MM, Maksimovic AZ, Tanuwidjaja I et al (2017) Characterization of enterococcal community isolated from an artisan Istrian raw milk cheese: biotechnological and safety aspects. Food Technol Biotechnol 55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17113/ftb.55.03.17.5118\u003c/span\u003e\u003cspan address=\"10.17113/ftb.55.03.17.5118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTsanasidou C, Asimakoula S, Sameli N et al (2021) Safety evaluation, biogenic amine formation, and enzymatic activity profiles of autochthonous enterocin-producing greek cheese isolates of the \u003cem\u003eEnterococcus faecium/durans\u003c/em\u003e Group. Microorganisms 9:777. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms9040777\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms9040777\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKiruthiga A, Padmavathy K, Shabana P et al (2020) Improved detection of \u003cem\u003eesp\u003c/em\u003e, \u003cem\u003ehyl\u003c/em\u003e, \u003cem\u003easa\u003c/em\u003e1, \u003cem\u003egel\u003c/em\u003eE, \u003cem\u003ecyl\u003c/em\u003eA virulence genes among clinical isolates of Enterococci. BMC Res Notes 13:170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13104-020-05018-0\u003c/span\u003e\u003cspan address=\"10.1186/s13104-020-05018-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEnnahar S (2000) Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol Rev 24:85\u0026ndash;106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0168-6445(99)00031-5\u003c/span\u003e\u003cspan address=\"10.1016/S0168-6445(99)00031-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFavaro L, Basaglia M, Casella S et al (2014) Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese. Food Microbiol 38:228\u0026ndash;239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fm.2013.09.008\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2013.09.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValledor SJD, Dioso CM, Bucheli JEV et al (2022) Characterization and safety evaluation of two beneficial, enterocin-producing \u003cem\u003eEnterococcus faecium\u003c/em\u003e strains isolated from kimchi, a Korean fermented cabbage. Food Microbiol 102:103886. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fm.2021.103886\u003c/span\u003e\u003cspan address=\"10.1016/j.fm.2021.103886\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrościańczyk A, Nowakiewicz A, Tracz AM, Bochniarz M (2025) Evaluation of the activity and molecular characterisation of bacteriocins produced by \u003cem\u003eE. faecium\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e isolated from different hosts against public health-threating pathogens. Microb Pathog 202:107432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2025.107432\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2025.107432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMancini L, Carbone S, Calabrese FM et al (2025) Isolation and characterization from raw milk of Enterocin B-producing \u003cem\u003eEnterococcus faecium\u003c/em\u003e: A potential dairy bio-preservative agent. Appl Food Res 5:100857. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.afres.2025.100857\u003c/span\u003e\u003cspan address=\"10.1016/j.afres.2025.100857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSonsa-Ard N, Rodtong S, Chikindas ML, Yongsawatdigul J (2015) Characterization of bacteriocin produced by \u003cem\u003eEnterococcus faecium\u003c/em\u003e CN-25 isolated from traditionally Thai fermented fish roe. Food Control 54:308\u0026ndash;316. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodcont.2015.02.010\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2015.02.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsaduzzaman SM, Sonomoto K (2009) Lantibiotics: Diverse activities and unique modes of action. J Biosci Bioeng 107:475\u0026ndash;487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jbiosc.2009.01.003\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiosc.2009.01.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNagao J (2009) Properties and applications of lantibiotics, a class of bacteriocins produced by Gram-positive bacteria. J Oral Biosci 51:158\u0026ndash;164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1349-0079(09)80024-8\u003c/span\u003e\u003cspan address=\"10.1016/S1349-0079(09)80024-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranz CMAP, Huch M, Abriouel H et al (2011) Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151:125\u0026ndash;140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2011.08.014\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2011.08.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u0026Ccedil;etin B, Aktaş H (2024) Monitoring probiotic properties and safety evaluation of antilisterial \u003cem\u003eEnterococcus faecium\u003c/em\u003e strains with cholesterol-lowering potential from raw Cow\u0026rsquo;s milk. Food Biosci 61:104532. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fbio.2024.104532\u003c/span\u003e\u003cspan address=\"10.1016/j.fbio.2024.104532\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmidi-Fazli N, Hanifian S (2022) Biodiversity, antibiotic resistance and virulence traits of \u003cem\u003eEnterococcus\u003c/em\u003e species in artisanal dairy products. Int Dairy J 129:105287. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.idairyj.2021.105287\u003c/span\u003e\u003cspan address=\"10.1016/j.idairyj.2021.105287\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u0026Ouml;zdemir R, Tuncer Y (2020) Detection of antibiotic resistance profiles and aminoglycoside-modifying enzyme (AME) genes in high-level aminoglycoside-resistant (HLAR) Enterococci isolated from raw milk and traditional cheeses in Turkey. Mol Biol Rep 47:1703\u0026ndash;1712. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11033-020-05262-4\u003c/span\u003e\u003cspan address=\"10.1007/s11033-020-05262-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArias CA, Murray BE (2012) The rise of the \u003cem\u003eEnterococcus\u003c/em\u003e: beyond vancomycin resistance. Nat Rev Microbiol 10:266\u0026ndash;278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro2761\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro2761\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCariolato D, Andrighetto C, Lombardi A (2008) Occurrence of virulence factors and antibiotic resistances in \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eEnterococcus faecium\u003c/em\u003e collected from dairy and human samples in North Italy. Food Control 19:886\u0026ndash;892. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodcont.2007.08.019\u003c/span\u003e\u003cspan address=\"10.1016/j.foodcont.2007.08.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCosta Y, Galimand M, Leclercq R et al (1993) Characterization of the chromosomal aac(6\u0026rsquo;)-Ii gene specific for Enterococcus faecium. Antimicrob Agents Chemother 37:1896\u0026ndash;1903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.37.9.1896\u003c/span\u003e\u003cspan address=\"10.1128/AAC.37.9.1896\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCharyyev MG, Tuncer B\u0026Ouml;, Kankaya DA, Tuncer Y (2019) Bacteriocinogenic properties and safety evaluation of \u003cem\u003eEnterococcus faecium\u003c/em\u003e YT52 isolated from boza, a traditional cereal based fermented beverage. J Consumer Prot Food Saf 14:41\u0026ndash;53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00003-019-01213-9\u003c/span\u003e\u003cspan address=\"10.1007/s00003-019-01213-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNami Y, Bakhshayesh RV, Jalaly HM et al (2019) Probiotic properties of \u003cem\u003eEnterococcus\u003c/em\u003e isolated from artisanal dairy products. Front Microbiol 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2019.00300\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.00300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFurlaneto-Maia L, Ramalho R, Rocha KR, Furlaneto MC (2020) Antimicrobial activity of enterocins against \u003cem\u003eListeria\u003c/em\u003e sp. and other food spoilage bacteria. Biotechnol Lett 42:797\u0026ndash;806. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10529-020-02810-7\u003c/span\u003e\u003cspan address=\"10.1007/s10529-020-02810-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDu R, Ping W, Ge J (2022) Purification, characterization and mechanism of action of enterocin HDX-2, a novel class IIa bacteriocin produced by \u003cem\u003eEnterococcus faecium\u003c/em\u003e HDX-2. LWT 153:112451. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2021.112451\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2021.112451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou C, Chang X, Zou Y et al (2024) The mechanism of \u003cem\u003eEnterococcus faecium\u003c/em\u003e on the virulence of \u003cem\u003eListeria monocytogenes\u003c/em\u003e during the storage of fermented sausages by whole genome analysis. Int J Food Microbiol 422:110826. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2024.110826\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2024.110826\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchittler L, Perin LM, Marques JL et al (2019) Isolation of \u003cem\u003eEnterococcus faecium\u003c/em\u003e, characterization of its antimicrobial metabolites and viability in probiotic Minas Frescal cheese. J Food Sci Technol 56:5128\u0026ndash;5137. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13197-019-03985-2\u003c/span\u003e\u003cspan address=\"10.1007/s13197-019-03985-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePopović N, Stevanović D, Radojević D et al (2023) Insight into the postbiotic potential of the autochthonous bacteriocin-producing \u003cem\u003eEnterococcus faecium\u003c/em\u003e BGZLM1-5 in the reduction in the abundance of \u003cem\u003eListeria monocytogenes\u003c/em\u003e ATCC19111 in a milk model. Microorganisms 11:2844. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms11122844\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms11122844\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoke H, Aslim B, Alp G (2010) The role of resistance to bile salts and acid tolerance of exopolysaccharides (EPSS) produced by yogurt starter bacteria. Arch Biol Sci 62:323\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2298/ABS1002323B\u003c/span\u003e\u003cspan address=\"10.2298/ABS1002323B\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta SBR, Sraboni FS, Naznin T et al (2025) Harnessing \u003cem\u003eEnterococcus faecium\u003c/em\u003e WFD-128 from yogurt fermentation: Unveiling probiotic attributes and targeted inhibition of \u003cem\u003eShigella sonnei\u003c/em\u003e diarrheal pathogenesis. Microb Pathog 204:107561. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2025.107561\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2025.107561\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnkaiah D, Esakkiraj P, Perumal V et al (2017) Probiotic characterization of \u003cem\u003eEnterococcus faecium\u003c/em\u003e por1: Cloning, over expression of Enterocin-A and evaluation of antibacterial, anti-cancer properties. J Funct Foods 38:280\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2017.09.034\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2017.09.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao J, Chen C, Fu Z et al (2024) Assessment of the safety and probiotic properties of \u003cem\u003eEnterococcus faecium\u003c/em\u003e B13 isolated from fermented Chili. Microorganisms 12:994. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms12050994\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms12050994\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhardwaj A, Gupta H, Kapila S et al (2010) Safety assessment and evaluation of probiotic potential of bacteriocinogenic \u003cem\u003eEnterococcus faecium\u003c/em\u003e KH 24 strain under in vitro and \u0026thinsp;in vivo\u0026thinsp;\u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;conditions. Int J Food Microbiol 141:156\u0026ndash;164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijfoodmicro.2010.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.ijfoodmicro.2010.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbedini R, Zaghari G, Jabbari L et al (2023) A potential probiotic \u003cem\u003eEnterococcus faecium\u003c/em\u003e isolated from camel rumen, fatty acids biotransformation, antilisteria activity and safety assessment. Int Dairy J 145:105706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.idairyj.2023.105706\u003c/span\u003e\u003cspan address=\"10.1016/j.idairyj.2023.105706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuan N, Liu L (2020) Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biotechnol 104:51\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-019-10226-1\u003c/span\u003e\u003cspan address=\"10.1007/s00253-019-10226-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh J, Metrani R, Shivanagoudra SR et al (2019) Review on bile acids: Effects of the gut microbiome, interactions with dietary fiber, and alterations in the bioaccessibility of bioactive compounds. J Agric Food Chem 67:9124\u0026ndash;9138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.8b07306\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.8b07306\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTilwani YM, Lakra AK, Domdi L et al (2022) Characterization of potential probiotic bacteria \u003cem\u003eEnterococcus faecium\u003c/em\u003e MC-5 isolated from the gut content of \u003cem\u003eCyprinus carpio\u003c/em\u003e specularis. Microb Pathog 172:105783. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.micpath.2022.105783\u003c/span\u003e\u003cspan address=\"10.1016/j.micpath.2022.105783\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmeida KV, Zanetti VC, Camelo-Silva C et al (2024) Powdered water kefir: Effect of spray drying and lyophilization on physical, physicochemical, and microbiological properties. Food Chem Adv 5:100759. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.focha.2024.100759\u003c/span\u003e\u003cspan address=\"10.1016/j.focha.2024.100759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbosa J, Borges S, Amorim M et al (2015) Comparison of spray drying, freeze drying and convective hot air drying for the production of a probiotic orange powder. J Funct Foods 17:340\u0026ndash;351. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2015.06.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2015.06.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang Y, Lin T, Zhu M, Yu S (2025) Complexation of Inulin with Proteins, Polysaccharides and Polyphenols: Their Interactions, Applications and Health Benefits. Food Reviews Int 41:1933\u0026ndash;1947. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/87559129.2025.2456544\u003c/span\u003e\u003cspan address=\"10.1080/87559129.2025.2456544\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKingwatee N, Apichartsrangkoon A, Chaikham P et al (2015) Spray drying \u003cem\u003eLactobacillus casei\u003c/em\u003e 01 in lychee juice varied carrier materials. LWT - Food Sci Technol 62:847\u0026ndash;853. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2014.12.007\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2014.12.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang S, Vignolles M-L, Chen XD et al (2017) Spray drying of probiotics and other food-grade bacteria: A review. Trends Food Sci Technol 63:1\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tifs.2017.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.tifs.2017.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRam\u0026iacute;rez-Dami\u0026aacute;n M, Garfias-Noguez C, Berm\u0026uacute;dez-Humar\u0026aacute;n LG, S\u0026aacute;nchez-Pardo ME (2025) Synbiotic microencapsulation of \u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;Lactobacillus\u0026thinsp;\u0026lt;\u0026thinsp;i\u0026thinsp;\u0026gt;\u0026thinsp;strains from mexican fermented beverages for enhanced probiotic functionality. Molecules 30:1185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules30051185\u003c/span\u003e\u003cspan address=\"10.3390/molecules30051185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReyes V, Chotiko A, Chouljenko A, Sathivel S (2018) Viability of \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e NRRL B-4495 encapsulated with high maize starch, maltodextrin, and gum arabic. LWT 96:642\u0026ndash;647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.lwt.2018.06.017\u003c/span\u003e\u003cspan address=\"10.1016/j.lwt.2018.06.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaseem Z, Mir SA, Wani SM et al (2025) Investigating gum arabic and soy protein isolate as wall material for encapsulation of five strains of \u003cem\u003eLactobacillus\u003c/em\u003e. Int J Biol Macromol 298:140083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2025.140083\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2025.140083\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarrar E, Mahdi AA, Sheth S et al (2021) Effect of maltodextrin combination with gum arabic and whey protein isolate on the microencapsulation of gurum seed oil using a spray-drying method. Int J Biol Macromol 171:208\u0026ndash;216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2020.12.045\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2020.12.045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoang NTN, Nguyen NNK, Nguyen LTK et al (2024) Research on optimization of spray drying conditions, characteristics of anthocyanins extracted from \u003cem\u003eHibiscus sabdariffa\u003c/em\u003e L. flower, and application to marshmallows. Food Sci Nutr 12:2003\u0026ndash;2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/fsn3.3898\u003c/span\u003e\u003cspan address=\"10.1002/fsn3.3898\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonstanty J, Tyrala D (2024) Particle sizing and surface area measurements: A comparative assessment of commercial air permeability and laser light diffraction instruments. Appl Sci 14:4802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app14114802\u003c/span\u003e\u003cspan address=\"10.3390/app14114802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFahrudin FI, Phongthai S, Intipunya P (2025) Enhancing stability of \u003cem\u003eBoesenbergia rotunda\u003c/em\u003e bioactive compounds: Microencapsulation via spray-drying and its physicochemical evaluation. Foods 14:2699. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods14152699\u003c/span\u003e\u003cspan address=\"10.3390/foods14152699\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaula DA, Martins EMF, Costa NA et al (2019) Use of gelatin and gum arabic for microencapsulation of probiotic cells from \u003cem\u003eLactobacillus plantarum\u003c/em\u003e by a dual process combining double emulsification followed by complex coacervation. Int J Biol Macromol 133:722\u0026ndash;731. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijbiomac.2019.04.110\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2019.04.110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmadi M, Khajeh F, Sohrabi S et al (2025) Spray-dried probiotic microcapsules with calcium cross-linked oxidized starch and inulin. Carbohydr Polym Technol Appl 10:100760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carpta.2025.100760\u003c/span\u003e\u003cspan address=\"10.1016/j.carpta.2025.100760\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeral HD, \u0026Ouml;zcan FŞ, \u0026Ouml;zcan N et al (2024) Determination of prebiotic activity and probiotic encapsulation ability of inulin type fructans obtained from \u003cem\u003eInula helenium\u003c/em\u003e roots. J Food Sci 89:5335\u0026ndash;5349. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1750-3841.17261\u003c/span\u003e\u003cspan address=\"10.1111/1750-3841.17261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;Amico V, Siepmann F, Siepmann J et al (2026) Microencapsulation of the probiotic \u003cem\u003eBifidobacterium longum\u003c/em\u003e by spray-drying: Formulation and process optimisation. J Drug Deliv Sci Technol 115:107670. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jddst.2025.107670\u003c/span\u003e\u003cspan address=\"10.1016/j.jddst.2025.107670\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYin M, Chen M, Yuan Y et al (2024) Encapsulation of \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GG in whey protein isolate-shortening oil and gum Arabic by complex coacervation: Enhanced the viability of probiotics during spray drying and storage. Food Hydrocoll 146:109252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodhyd.2023.109252\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2023.109252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacteriocins, Functional Products, Lactic Acid Bacteria, Microencapsulation, Spray-drying","lastPublishedDoi":"10.21203/rs.3.rs-8297099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8297099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotics have attracted increasing interest from consumers due to their potential health benefits, antimicrobial properties, and overall wellness support. In this context, it is essential to identify new bacterial strains that exhibit significant functional characteristics and enhanced stability to food processing. This study aimed to identify and assess the technological and probiotic properties of \u003cem\u003eEnterococcus faecium\u003c/em\u003e EM03, which was isolated from semi-hard cheese commercialized in Barreiras, Bahia, Brazil. The isolate was genotypically confirmed as \u003cem\u003eE. faecium\u003c/em\u003e through \u003cem\u003e16S rRNA\u003c/em\u003e gene sequencing. \u003cem\u003eIn vitro\u003c/em\u003e safety assessments revealed sensitivity to most of the antibiotics tested, with resistance observed only to ciprofloxacin and gentamicin. Molecular analyses indicated the absence of genes associated with the production of biogenic amines and several virulence factors. Conversely, the presence of genes linked to bacteriocin production, specifically \u003cem\u003eent\u003c/em\u003eB, \u003cem\u003eent\u003c/em\u003eP, L50AB, \u003cem\u003eIan\u003c/em\u003eM, and \u003cem\u003eIan\u003c/em\u003eC, was confirmed. These findings were further supported by results from the spot-on-the-lawn assay, which demonstrated that \u003cem\u003eE. faecium\u003c/em\u003e EM03 inhibited the growth of \u003cem\u003eListeria monocytogenes\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e through the production of proteinaceous compounds. Regarding its probiotic potential, the isolate exhibited high survival rates under simulated gastrointestinal conditions, including tolerance to pH 3.5, to pepsin treatment, and to bile salts. Additionally, microencapsulation by spray-drying allowed the survival to \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal tests and preserved probiotic viability for up to 60 days, with stable physical properties of the microparticles. Overall, the results indicate \u003cem\u003eE. faecium\u003c/em\u003e EM03 has technological potential for food and health-related applications.\u003c/p\u003e","manuscriptTitle":"Probiotic and technological potential of Enterococcus faecium EM03, isolated from Brazilian semi-hard cheese","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 09:36:33","doi":"10.21203/rs.3.rs-8297099/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-18T13:52:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T08:49:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T14:04:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-03T14:14:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217401432601323343021718031870922552277","date":"2026-01-20T15:30:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T12:33:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173500637027907763004751981567151366099","date":"2025-12-12T08:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89403635070760341718457949942308474001","date":"2025-12-12T05:54:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219582252608748352078938373879635851451","date":"2025-12-11T18:28:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149157670850575504034818001393612454325","date":"2025-12-11T16:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T15:50:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T02:44:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T02:44:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-12-06T23:44:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26acb03d-f199-4c6c-9d7a-ffb419b5c9ac","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T14:55:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 09:36:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8297099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8297099","identity":"rs-8297099","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}