Dissecting the Genetic Basis of the Technological, Functional, and Safety Characteristics of Lacticaseibacillus paracasei SRX10.

Nonstarter lactic acid bacteria (NSLAB) are major contributors to the unique characteristics (e.g., aroma, flavor, texture) of dairy and nondairy fermented products. Lc. paracasei SRX10 is an NSLAB strain originally isolated from a traditional Greek cheese and previously shown to exhibit favorable biotechnological characteristics. More specifically, the strain showed tolerance to simulated gastrointestinal conditions, exopolysaccharide (EPS) biosynthetic capacity, and lack of hemolytic activity and was used in the production of yoghurt and feta cheese with distinct organoleptic characteristics. The aim of the present study was to investigate these traits at the genome level through whole-genome sequencing (WGS), annotation, and comparative genomics. Functional annotation of the genome revealed that Lc. paracasei SRX10 can utilize different carbon sources, leading to the generation of flavor compounds, including lactic acid, acetate, ethanol, and acetoin. Similarly, full clusters for fatty acid biosynthesis, protein and peptide degradation, as well as genes related to survival under extreme temperatures, osmotic shock, and oxidative stress were annotated. Importantly, no transferable antibiotic resistance genes or virulence factors were identified. Finally, strain-specific primers based on genome-wide polymorphisms were designed for the efficient and rapid identification of Lc. paracasei SRX10 via multiplex PCR in fermented products.

Application of multi-functional lactic acid bacteria strains in a pilot scale feta cheese production.

Feta cheese is the most recognized Greek Protected Designation of Origin (PDO) product in the world. The addition of selected autochthonous lactic acid bacteria (LAB) strains to cheese milk as adjunct cultures is gaining more attention, since they can impact the nutritional, technological and sensory properties of cheeses, as well as improve the safety of the product. The aim of this study was to produce Feta cheese with enhanced quality and safety, and distinctive organoleptic characteristics by applying autochthonous lactic acid bacteria (LAB) with multi-functional properties as adjunct cultures. Feta cheeses were produced with the commercial lactococcal starter culture and the addition of 9 LAB strains (Lactococcus lactis SMX2 and SMX16, Levilactobacillus brevis SRX20, Lacticaseibacillus paracasei SRX10, Lactiplantibacillus plantarum FRX20 and FB1, Leuconostoc mesenteroides FMX3, FMX11, and FRX4, isolated from artisanal Greek cheeses) in different combinations to produce 13 cheese trials (12 Feta trials with the adjunct LAB isolates and the control trial). In addition, Feta cheese manufactured with FMX3 and SMX2 and control Feta cheese were artificially inoculated (4 log CFU/g) with Listeria monocytogenes (a cocktail of 4 acid or non-acid adapted strains). Cheese samples were monitored by microbiological and physicochemical analyses during ripening, and microbiological, physicochemical, molecular and sensory analyses during storage at 4°C. The results showed that after manufacture, the LAB population was ca. 9.0 log CFU/g at all samples, whereas during storage, their population declined to 6.5-7.0 log CFU/g. In the Listeria inoculated samples, Listeria was absent after 60 days (end of ripening) and after 90 days in the adjunct culture, and in the control trials, respectively. Moreover, the addition of selected strains, especially Lcb. paracasei SRX10, led to cheeses with desirable and distinctive organoleptic characteristics. Furthermore, randomly amplified polymorphic PCR (RAPD-PCR) molecular analysis confirmed that the multi-functional LAB strains were viable by the end of storage. Overall, the results of this study are promising for the use of autochthonous strains in various combinations with the commercial starter culture to satisfy industry requirements and consumer demands for traditional and high added value fermented products.

Development of a Multiplex PCR Assay for Efficient Detection of Two Potential Probiotic Strains Using Whole Genome-Based Primers.

Probiotics are microorganisms that exert strain-specific health-promoting effects on the host. Τhey are employed in the production of functional dairy or non-dairy food products; still, their detection in these complex matrices is a challenging task. Several culture-dependent and culture-independent methods have been developed in this direction; however, they present low discrimination at the strain level. Here, we developed a multiplex PCR assay for the detection of two potential probiotic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum L125 and Lp. pentosus L33, in monocultures and yogurt samples. Unique genomic regions were identified via comparative genomic analysis and were used to produce strain-specific primers. Then, primer sets were selected that produced distinct electrophoretic DNA banding patterns in multiplex PCR for each target strain. This method was further implemented for the detection of the two strains in yogurt samples, highlighting its biotechnological applicability. Moreover, it can be applied with appropriate modifications to detect any bacterial strain with available WGS.

Role of Microbial Interactions across Food-Related Bacteria on Biofilm Population and Biofilm Decontamination by a TiO2-Nanoparticle-Based Surfactant.

Microbial interactions play an important role in initial cell adhesion and the endurance of biofilm toward disinfectant stresses. The present study aimed to evaluate the effect of microbial interactions on biofilm formation and the disinfecting activity of an innovative photocatalytic surfactant based on TiO2 nanoparticles. Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, Leuconostoc spp., Latilactobacillus sakei, Serratia liquefaciens, Serratia proteomaculans, Citrobacter freundii, Hafnia alvei, Proteus vulgaris, Pseudomonas fragi, and Brochothrix thermosphacta left to form mono- or dual-species biofilms on stainless steel (SS) coupons. The effectiveness of the photocatalytic disinfectant after 2 h of exposure under UV light on biofilm decontamination was evaluated. The effect of one parameter i.e., exposure to UV or disinfectant, was also determined. According to the obtained results, the microbial load of a mature biofilm depended on the different species or dual species that had adhered to the surface, while the presence of other species could affect the biofilm population of a specific microbe (p < 0.05). The disinfectant strengthened the antimicrobial activity of UV, as, in most cases, the remaining biofilm population was below the detection limit of the method. Moreover, the presence of more than one species affected the resistance of the biofilm cells to UV and the disinfectant (p < 0.05). In conclusion, this study confirms that microbial interactions affected biofilm formation and decontamination, and it demonstrates the effectiveness of the surfactant with the photocatalytic TiO2 agent, suggesting that it could be an alternative agent with which to disinfect contaminated surfaces.

Mapping the Key Technological and Functional Characteristics of Indigenous Lactic Acid Bacteria Isolated from Greek Traditional Dairy Products.

The aim of the current study was to isolate indigenous lactic acid bacteria (LAB) from traditional Greek cheeses and assess their biochemical, technological, and functional characteristics, so as to develop novel cultures with multi-functional properties. Hence, 109 LAB isolates were recovered from traditional fresh cheeses and were evaluated in vitro for their gas production; proteolytic, lipolytic, and haemolytic activity; exopolysaccharide production (EPS); enzymatic potential; and ability to grow at 6.5% NaCl and at different pH, temperature, and anaerobic conditions. Consequently, 48 selected isolates were further evaluated for their survival under simulated gastrointestinal tract conditions, partial bile salt hydrolase activity, antibiotic resistance, and antimicrobial activity against pathogens. These isolates were also incorporated as co-cultures in yogurt production to examine their sensory characteristics and their survival in the product. Some prominent isolates that showed favorable technological and functional characteristics (good survival rates at low pH and bile salts, ability to produce ß-galactosidase, and EPS) and attributed desirable sensory characteristics to yogurt were Lactococcuslactis (SRX2, SRX3, SRX5, and SMX16), Lactobacillus paracasei SRX10, and Lactiplantibacillusplantarum (FRX7, FB1), while Leuconostoc mesenteroides FMX3 and L. lactis SMX2 showed an anti-listerial activity in vitro. The results of the present study are promising for the production of novel dairy functional products with an enhanced quality and safety.