1. INTRODUCTION
Enterococci are a group of lactic acid bacteria commonly found in various food ingredients, including vegetables, fruits, raw milk, and dairy products like cheese and cured meats [1,2]. Among these, species such as Enterococcus faecium (E. faecium), Enterococcus durans, and Enterococcus lactis are particularly promising as biological products due to their probiotic properties, including their ability to produce bacteriocins with antibacterial effects [1,3,4]. Several Enterococci strains have been used as effective probiotics in clinical settings for many years [5,6]. For example, E. faecium strains such as SF68, M74, LX, and L3 have been demonstrated in multiple randomized clinical studies to be particularly effective in treating gastrointestinal diseases, including chronic gastritis, gastric ulcers, irritable bowel syndrome, pancreatitis, and chronic hepatitis [7]. The E. lactis has been recently studied and separated from Group B of E. faecium with less pathogenic potential [8]. Although the use of E. lactis as a commercial product is currently limited, its safety and probiotic properties have been investigated, showing promise for future probiotic applications [9].
Fermented foods and drinks, whether derived from animal or plant sources, play a crucial role in our diets. These foods typically contain lactic acid bacteria, which thrive during the fermentation process [10,11]. Lactic acid bacteria naturally produce compounds such as organic acids, ethanol, and antimicrobial substances that inhibit spoilage organisms and pathogenic bacteria in fermented foods [12]. Moreover, these bacteria are well-adapted to spontaneous fermentation and contribute significantly to the health of both humans and animals, particularly in the digestive tract, where they function as probiotics. Therefore, fermented foods are considered rich sources for isolating probiotics [10,11].
Fermented pork roll (nem chua) is a traditional Vietnamese food from which many probiotics have been isolated. According to previous studies, probiotic strains isolated from fermented pork rolls belong to the Lactobacillus, Lactococcus, and Pediococcus groups [13]. Stinky tofu is a special food in the Asian region that originated from China. Similar to other fermented foods, stinky tofu is also a rich source of probiotics such as Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, Pediococcus, Streptococcus, and Weissella [14].
Previous studies have often isolated common probiotics such as Lactobacillus and Lactococcus from stinky tofu and fermented pork roll. Although Enterococcus bacteria are present, they were rarely isolated and studied. Therefore, in this study, four Enterococcus strains were isolated and analyzed for their properties. These strains were identified through gene sequencing, and their safety and probiotic properties were investigated. This study identified promising bacterial strains and provided valuable insights for the development of health-promoting probiotic products.
2. MATERIALS AND METHODS
2.1. Food materials
Samples of fermented foods, including stinky tofu and Vietnamese fermented pork rolls were collected from traditional markets in Ha Noi–Vietnam, in 2022.
2.2. Bacteria strains
Bacteria strains including Escherichia coli American Type Culture Collection (ATCC) 25922, Shigellasonnei (levine) Weldin ATCC 25931, Staphylococcus aureus ATCC 25923, and Salmonella enterica Typhimurium ATCC 14028 were purchased from ATCC. Listeriamono cytogenes SLR2249 was provided by Hardy Diagnostics Company.
2.3. Probiotics isolation
All food materials were crushed and immersed in a physiological saline solution (0.9% NaCl). Bacterial cells in the liquid suspension were cultured on HiCrome™ E. faecium Agar medium (Himedia) for 24 hours at 37°C [10]. Subsequently, target cells (green-colored colonies along with yellow coloration to the medium) were streaked to Chromatic detection agar (Liofilchem) and incubated at 37?C for 24 hours. Then, green colonies were cultured on Lactobacillus de Man, Rogosa and Sharpe (MRS) Agar (Himedia) before species identification. Next, bacteria were suspended in a sodium chloride 0.45% solution to attain a density of 0.5–0.63 McF and identified using the VITEK® 2 compact system with GP Card (BioMerieux).
2.4. Genome sequencing
Total DNA was extracted utilizing the DNeasy Blood & Tissue Kits (Qiagen). Subsequently, 2 × 150 bp paired-end libraries were prepared employing the Nextera DNA Sample Preparation Kit (Illumina Inc., United States) following the manufacturer’s instructions. Then, genome sequencing was executed using the Illumina HiSeqXten sequencing 150PE platform (Illumina Inc., United States). The quality assessment of raw sequencing data was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and low-quality and short-length reads were filtered out by trimmomatic [15]. The high-quality pair-end reads data was de-novo assembled by SPAdes (Galaxy version 3.12.0) [16]. The completeness of a draft assembly was assessed using BUSCO scores [17].
2.5. Species identification
The assembled genome sequences underwent ribosomal multilocus sequence typing (rMLST) analysis using the online database and platform (available at https://www.pubmlst.org/species-id) [18].
2.6. Phylogenetic analysis
Gene identifications were automatically done using Prokka [19] and via aligning scaffolds to target sequences with minimap2 [20]. The Phylogenetic analysis was conducted utilizing the Molecular Evolutionary Genetic Analysis (MEGA X) software [21].
2.7. Antibiotic susceptibility
The minimum inhibitory concentration (MIC) results were generated using the Vitek 2 system with AST-GP67 test cards (bioMérieux) following the manufacturer’s instructions. Antibiotic susceptibility was automatically assessed by this system according to Clinical Laboratory Standards Institute guidelines and natural resistance patterns.
2.8. Hemolytic activity
The hemolytic activity of bacteria was assessed following the protocol outlined by Angmo et al. [22]. Briefly, the bacteria were cultured on Columbia Agar Base (Himedia) supplemented with 5% Sheep Blood for 48 hours at 37°C.
2.9. Genotypic virulence determinants
The presence of virulence factors, biogenic amines, and antibiotic resistance genes was assessed through polymerase chain reaction (PCR) using DreamTaq green PCR master mix (Thermos Scientific) and gene-specific primers (Table 1). The PCR products were then visualized by electrophoresis on 2% agarose gels.
2.10. Acid and bile salt tolerance
Bacteria cultured in MRS (De Man – Rogosa – Sharpe) medium were harvested and washed with a 0.45% sodium chloride solution by centrifugation at 5,000 g for 10 minutes. The cell pellets were used for assessments.
For acid tolerance assessment, the cell pellets were re-suspended in an MRS liquid medium adjusted to pH 2.5, aiming for a density of 0.5–0.63 McF. These bacterial suspensions were then incubated for 0, 2, and 4 hours at 37°C. Following this treatment, the suspensions were spread onto MRS solid medium and incubated for 24 hours at 37°C. Surviving cells were quantified based on the number of colonies formed [23].
For the assessment of bile salt tolerance, the procedure was conducted similarly to the method described above. However, in this case, bacterial cells were treated with MRS liquid medium supplemented with either 0.3% or 1% ox-bile for 0 or 4 hours. To assess simultaneous acid and bile salt resistance, bacterial cells were treated with MRS liquid medium at pH 2.5, supplemented with 0.3% or 1% bile salts.
2.11. Antimicrobial activity
Antimicrobial activity was assessed following the method described by Nami et al. [31]. Indicator bacteria were initially cultured on MRS agar at 37°C for 24 hours. The bacteria were then suspended in water to a concentration of 1.5 × 108 CFU/ml and spread onto MRS agar plates. Wells with a diameter of 5 mm were then created in these plates. Subsequently, 50 μl of filtered cell-free supernatant obtained from the cultures of the isolates, with a cell density of 108 CFU/ml, was added to each well and allowed to diffuse for 4 hours at room temperature. After 24 hours of incubation at the optimal growth temperature of the indicator strains, the inhibition zones around the wells were measured using a digital caliper. Experiments were performed in triplicate, with three plates per replicate.
2.12. Cholesterol assimilation
The cholesterol assimilation was determined using the o-phthalaldehyde method described by Usman and Hosono [32] and Asan-Ozusaglam and Gunyakti [33] with some modifications. Briefly, the bacteria were cultured in MRS broth supplemented with 0.3% ox gall (Merk, Germany) and cholesterol (150 µg/ml; Sigma-Aldrich) at 37°C for a day. After incubation, the cells were removed by centrifugation at 10,000 g for 15 minutes. Subsequently, a mixture consisting of 1 ml of cell-free broth, 1 ml of KOH (33% w/v), and 2 ml of 96% ethanol was heated at 60°C for 15 minutes. After cooling to room temperature, 2 ml of water and 3 ml of hexane were added and mixed for 1 minute. One ml of the hexane layer was transferred into a glass tube and evaporated in a water bath at 80°C. The residue was dissolved in 2 ml of 0.05% (w/v) o-phthalaldehyde reagent (Merck, Germany). After standing for 10 minutes, 0.5 ml of concentrated sulfuric acid was added. The absorbance was measured using a spectrophotometer at 550 nm.
 | Table 1. List of primers used in this study. [Click here to view] |
Cholesterol assimilation was calculated using the following equation: A = 100−[(B/C) × 100], where A represents the percentage of cholesterol assimilation (%), B is the amount of cholesterol in the inoculated medium, and C is the amount of cholesterol in the non-inoculated (control) medium.
2.13. Statistical analysis
Statistical analysis was performed using one-way ANOVA, followed by Duncan’s multiple range tests, in SPSS software (version 20). Statistical significance was set at p < 0.05. Data are presented as mean ± standard deviation, based on three biological replicates.
 | Table 2. Isolation of Enterococcus strains in traditional fermented foods. [Click here to view] |
3. RESULTS
3.1. Isolation of Enterococcus strains from traditional fermented foods
To isolate probiotics, samples were collected from two sources of stinky tofu and two sources of fermented pork rolls. Bacterial suspensions from these foods were cultured and selected on E. faecium HiCrome™ agar, Chromatic detection agar, and Lactobacillus MRS agar. As a result, four bacterial strains including F20BA, F26BA, F53BA, and F54BA were isolated, each corresponding to a different food sample (Table 2). The F20BA and F26BA strains were isolated from fermented pork rolls, while the F53BA and F54BA strains were isolated from stinky tofu samples. The identification results using the VITEK® 2 system indicated that all four strains were identified as E. faecium with accuracy ranging from 96% to 98% (data not shown).
3.2. Species identification of new isolates
Some Enterococcus species are closely related, making them difficult to distinguish using conventional taxonomic methods [34,35]. Therefore, analysis of rMLST and Rhomboid protease (GluP) gene sequences was utilized for species identification.
 | Supplementary Table 1. Genome assembly of bacteria strains. [Click here to view] |
 | Supplementary Table 2. Species identification by rMLST analysis. [Click here to view |
Whole-genome sequencing and MLST analysis had demonstrated efficacy in species identification of Enterococcus spp [35]. In this study, genome sequencing of four isolated strains was conducted utilizing the Illumina Next-Generation Sequencing platform. More than 10.5 million qualified reads were obtained. The assembled genome sequences ranged from 2.63 to 2.77 Mb in length, with coverage between 533 and 599. Genome completeness was from 98.4% to 99.2% (Supplementary Table 1). Species identifications were performed using rMLST analysis with the assembled genome sequences. Analysis of data from 55 genes encoding bacterial ribosome protein subunits revealed that all four isolates were Enterococcuslactis (Table 2, Supplementary Table 2).
The GluP has been identified as the best candidate gene for distinguishing between E. faecium and E. lactis [36]. In this study, GluP sequences from isolated strains, retrieved from genome sequencing data, were compared with their orthologs in the Enterococcus genome using phylogenetic analysis (Fig. 1). The analysis revealed that all four GluP sequences from the isolates exhibited high similarity with those of E. lactis and showed distinct distances from other orthologous genes. This result is consistent with the rMLST analysis, which also identified all four isolates as E. lactis.
 | Figure 1. Dendrogram illustrating the relationship between GluP gene sequences from isolated strains and Enterococcus spp. The tree was constructed by MEGA X using maximum likelihood method and JTT matrix-based model. Numbers at nodes indicate the percentage bootstrap scores from 10,000 replicates. The scale bar represents 0.05 estimated number of substitution events per site. [Click here to view] |
 | Figure 2. Hemolytic assays of isolated strains. The bacteria was grown on Columbia Agar supplemented with 5% sheep blood for 24 hours at 37?C. Scale bar: 1 cm [Click here to view] |
3.3. Safety assessment of isolated strains
3.3.1. Hemolytic activity
The bacteria were cultured on Columbia Agar with 5% Sheep Blood to assess their hemolytic capacity. After 24 hours of incubation, no discernible change was observed in the medium under and around the colonies (Fig. 2). Consequently, all four isolates did not induce hemolysis. This result suggests that the isolates are non-hemolytic or exhibit γ-hemolysis.
3.3.2. Virulence factors
The presence of common virulence factors in Enterococcus genomes was assessed via PCR analysis. Consequently, the virulence-encoding genes including Enterococcal surface protein (Esp), serine protease (SprE), fsrB, surface aggregating protein (asa1), cytolysin A (cylA), cytolysinM (CylM), as well as the bacterial toxin-encoding genes Histidine decarboxylase (Hdc1, Hdc2), Tyrosine decarboxylase (Tdc) were negative for PCR (Table 3). Furthermore, these coding sequences were found to be absent in the assembled genome (data not shown).
3.3.3. Antibiotic susceptibility
Antibiotic susceptibility is a key criterion for evaluating the safety of probiotics. In this study, antibiotic susceptibility was assessed using the Vitek 2 Compact system. As a result, all bacterial strains were susceptible to at least 10 of the 13 antibiotics used (Table 4). All strains showed moderate to high resistance to erythromycin. But, only F20BA and F54 are resistant to tetracycline, and F26BA and F53BA are resistant to nitrofurantoin. Notably, all four strains were sensitive to vancomycin.
3.4. Determination of probiotic potential
3.4.1. Acid and bile salt tolerance
Acid tolerance of the strains was assessed by incubating bacteria in an MRS medium with a pH of 2.5 for 1 to 4 hours. The results showed that, except for F54BA, which had a survival rate between 88.7% ± 0.3% and 92.2% ± 0.4%, the other three strains demonstrated higher tolerance, with survival rates exceeding 95.5% (Table 5).
To assess bile salt tolerance, bacteria were incubated in MRS medium containing either 0.3% or 1% bile salt. The probiotic strains continued to survive and grow in both bile salt concentrations, with survival rates ranging from 84% to 99.2% (Table 5). In 0.3% bile salt, strains F20BA and F53BA demonstrated higher survival rates (98.2% to 99.2%) compared to the other two strains, which ranged from 85.3% to 90%. Notably, strain F53BA maintained high tolerance in 1% bile salt, with survival rates of approximately 98.2% to 98.3%. These results indicate that F53BA exhibits better bile salt tolerance than the other strains.
The combined effect of acid and bile salt on bacterial survival was also investigated. Strain viability was assessed in MRS medium (pH 2.5) supplemented with 0.3% and 1% bile salt. The results showed that in both 0.3% and 1% bile salt environments, the probiotic strains continued to survive and grow, with a survival ratio ranging from 85% to 99.2% (Table 5). In particular, the survival rate of strain F54BA (85% to 88.8%) was lower compared to the other three strains, which had survival rates ranging from 89.9% to 99.2%.
3.4.2. Antimicrobial activity
Four bacterial strains were co-cultured with other pathogenic microorganisms capable of transmission through the human digestive tract, including E. coli, Salmonella, Shigella, Staphylococcus aureus, and Listeria. After 24 hours of co-cultivation, inhibition zones emerged surrounding all wells (Table 6). These findings indicate that all four isolated strains effectively suppressed the growth of the tested bacterial pathogens. Remarkably, all four strains inhibit Escherichia coli, Salmonella, and Shigella more effectively as evidenced by the presence of larger inhibition zones exceeding 20 mm in diameter. Additionally, F26BA exhibited superior effectiveness in inhibiting all five tested pathogens among the isolates.
 | Table 3. PCR detection of virulence factors, vancomycin resistance genes, and biogenic amines encoding genes. [Click here to view] |
 | Table 4. Antibiotic susceptibility of isolated strains. [Click here to view] |
3.4.3. Cholesterol assimilation
To assess cholesterol assimilation, bacterial strains were cultured in a medium containing cholesterol. After 24 hours of incubation, varying degrees of reduction in cholesterol levels were observed, ranging from 10% to 38% (Fig. 3). Therefore, all four strains have cholesterol assimilation ability. The F20BA strain exhibited the highest assimilation rate at 38.8% ± 0.3%. Subsequently, strains F26BA and F54BA followed with assimilation rates of 24.5% ± 1.4% and 20.8% ± 0.6%, respectively. In contrast, the F53BA strain displayed the lowest assimilation ability, recording only 10% ± 1%.
 | Table 5. Viability of isolated E. lactis strains after exposure to low pH and bile salt. [Click here to view |
 | Table 6. Antimicrobial activity of isolated strains against pathogenic microorganisms. [Click here to view] |
 | Figure 3. Cholesterol assimilation ability of isolated strains. Data are presented as mean ± SD of three replicates. Different letters indicate significant differences based on one-way ANOVA analysis with Duncan’s test (p < 0.05). [Click here to view] |
4. DISCUSSION
4.1. Four E. lactis trains from local traditional fermented foods
Certain Enterococcus species pose challenges in differentiation through conventional methods due to their close genetics [35]. In this study, Enterococcus strains were isolated from local fermented foods using a specific selection medium, and species identification was conducted utilizing rMLST. Consequently, these strains were identified as belonging to E. lactis (Supplementary Table 2). The E. lactis was proposed as an independent species in 2012 [2], although the earlier strains were isolated from milk samples [37]. Within the Enterococcus genus, lactis and faecium are closely related species. Recently, based on genome studies, clade B of E. faecium has been proposed to be reclassified as E. lactis [8]. gluP was identified as the most promising candidate for distinguishing between these two species [36]. The results indicate that the gluP gene sequences of all four strains exhibit high similarity when compared to those of E. lactis (Fig. 1). The data presented above demonstrate that the isolated strains belong to E. lactis.
The E. lactis is a significant probiotic strain that has been isolated from diverse sources. Among these, fermented foods stand out as rich reservoirs of lactic acid bacteria. Similar to this study, numerous strains have been recovered from this source, including those found in dairy products and rice wine koji [38], radish pickle fermentation [39], and raw milk cheeses [2]. Besides, E. lactis has also been isolated from other sources such as human gut [31,38,40], and raw shrimps [41]. However, studies on isolating E. lactis from fermented foods are limited. This study is the first to report the isolation and evaluation of E. lactis in Vietnamese fermented pork rolls and local stinky tofu.
4.2. Probiotic potential of isolates
In addition to being used as probiotics, some Enterococci strains are known to be pathogenic and can cause clinical diseases such as such as bacteremia, infectious endocarditis, and urinary tract infections [42,43]. Therefore, safety is the primary requirement for Enterococcus spp. strains intended for probiotic production [44]. The strains must be susceptible to key antibiotics used in treating intestinal pathogens such as vancomycin, should not contain genes expressing major virulence factors such as cylA, cylB, cylM, esp, and gelE, and must not be hemolytic [9,31,45]. Moreover, biogenic amines produced by Enterococci are known for their harmful effects on human health [46]. Therefore, selecting Enterococcus strains that lack major virulence factors, are vancomycin-susceptible, and are incapable of producing biogenic amines has become a popular procedure for their use as probiotics [31,46–48]. In this study, all four isolated strains tested negative for PCR detection of vancomycin resistance genes (VanA, VanB), virulence factor encoding genes (Esp, SprE, fsrB, asa1, CylA, CylM) and biogenic amine metabolism genes (Hdc1, Hdc2, Tdc). Additionally, they are sensitive to numerous antibiotics, including vancomycin (Table 4), and are non-hemolytic (Fig. 2). Furthermore, compared to E. faecium, E. lactis strains also cause human infections, but to a much lesser extent [36,38]. Based on genetic analysis, E. lactis contains fewer antibiotic-resistance genes than E. faecium [38] and lacks hospital infection-associated markers [8]. These data indicate the basic safety parameters of the isolates.
In this study, the isolated strains showed inhibitory effects on enteropathogenic bacteria including E. coli, Salmonella, Shigella, Staphylococcus aureus, and Listeria (Table 6). These abilities of E. lactis were also reported in previous studies [9,41,49]. Additionally, these strains can survive and grow in bile and low-pH environments (Table 5). These properties are essential for bacteria to thrive in the gut and compete with other microbial species [46]. In addition, the ability to assimilate cholesterol provides the potential for developing cardiovascular support products, especially isolated strain F20BA.
In conclusion, the initial in-vitro tests in this study provided preliminary evidence that the isolated bacterial strains have potential as probiotics. However, additional studies are necessary to progress toward commercial applications. For instance, the studies include assessing the activities of isolated strains in simulated gastrointestinal environments and conducting clinical trials.
5. CONCLUSION
In this study, two E. lactis strains, F20BA and F26BA, were isolated from fermented pork rolls, while two other strains, F53BA and F54BA, were isolated from stinky tofu. Those are vancomycin susceptible, lacking common virulence-encoding genes, and non-hemolytic bacteria. Besides, they exhibit potential probiotic properties such as acid and bile salt tolerance, antimicrobial activity, and cholesterol assimilation. However, further studies are needed to support the development of new probiotic products from these strains.
6. SUPPLEMENTARY INFORMATION
Supplementary Table 1: Genome assembly of bacteria strains.
Supplementary Table 2: Species identification by rMLST analysis.
7. AUTHORS’ CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. FUNDING
This research was supported by project “Investigating the production of probiotics from Enterococcus spp” and by the Joint Vietnam-Russia Tropical Science and Technology Research Center. Funding was provided by The General Department of Logistics—Ministry of National Defense (Vietnam).
9. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
10. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
11. DATA AVAILABILITY
All the data are available with the authors and shall be provided upon reasonable request. The genome sequencing data have been deposited in GenBank (NCBI) under the following accession numbers: JARWSG000000000 (F20BA), JARGGM000000000 (F26BA), JARJOX000000000 (F53BA), and JARDYX000000000 (F54BA).
12. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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