Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 μg/mL) and vancomycin (MIC = 2 μg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry

Garlic, onion, and cinnamon essential oil anti-biofilms' effect against Listeria monocytogenes

Biofilms represent a serious problem for food industries due to their persistence in processing surfaces, from which they can cause food spoilage or, even worse, lead to foodborne diseases. Microorganisms immersed in biofilms are more resistant to biocides. The search for natural effective alternatives for the prevention and the control of biofilms has increased lately. The aim of this research was to test the antibacterial and the anti-biofilm activities of cinnamon, onion, and garlic essential oils againstListeria monocytogenes. The methodology highlighted first the effect of these essential oils onL. monocytogenesusing disc diffusion and minimum inhibitory concentration (MIC) methods and then on initial cell attachment and six hours preformed biofilms. The inhibition of biofilms was assessed by crystal violet assay. Sulfides were the most abundant compounds present in onion and garlic essential oils, while cinnamaldehyde was predominant in cinnamon essential oil. MIC values were of 0.025 mg mL(-1)for onion essential oil and 0.100 mg mL(-1)for cinnamon and garlic. Onion essential oil inhibited initial cell attachment by 77% at 0.5 of the MIC dose, while at MIC, cinnamon and garlic essential oils inhibited the initial microbial adhesion completely. All three essential oils completely inhibited initial cell attachment when applied at 2 MIC. On the contrary, preformed biofilms were more resistant, and the inhibition rate ranged from 33% to 78%. In summary, this investigation revealed that the essential oils of garlic, onion, and cinnamon show an effective antibiofilm activity againstL. monocytogenesand are promising natural antimicrobial alternatives for food processing facilities

<i>Lactiplantibacillus plantarum</i> from Unexplored Tunisian Ecological Niches: Antimicrobial Potential, Probiotic and Food Applications

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0–6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices

Multi-Functional Potential of Lactic Acid Bacteria Strains and Antimicrobial Effects in Minimally Processed Pomegranate (<i>Punica granatum</i> L. cv Jolly Red) Arils

This study aimed to evaluate the antimicrobial activity of both cells, and cell-free supernatants (CFS) of 7 selected lactic acid bacteria (LAB) strains belonging to Limosilactobacillus fermentum (4 strains), Lacticaseibacillus paracasei (1 strain), Lacticaseibacillus rhamnosus (1 strain), and Enterococcus faecium (1 strain) species, against Listeria monocytogenes, Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus, by both the agar-well diffusion and co-culture methods. In addition, probiotic and safety traits were also detected. Great variability was detected on antimicrobial effects, whereas all tested strains were found sensitive to most of the tested antibiotics, and without any DNase, gelatinase, or hemolytic activity. Moreover, strains showed excellent survival in acidic conditions and exhibited tolerance to pepsin and bile salts. Based on the in vitro results, the CFSs of two selected L. fermentum strains were applied, in a mixed solution, as bio-preservative into minimally processed pomegranate arils, inoculated with a cocktail of L. monocytogenes and E. coli. Samples, packaged in an ordinary atmosphere, were analyzed during refrigerated storage, for up to 12 days, for physicochemical (as weight loss, texture, color, pH, total soluble solids and organic acid content) and for microbiological traits. Results revealed the effectiveness of CFS, up to 12 days, in reducing weight loss and microbial growth, without any significant effect on texture, total soluble solid content and color, found comparable to the acid citric treatment, highlighting the multi-functional potential of selected probiotic strains