Novel Probiotic Candidates in Artisanal Feta-Type Kefalonian Cheese: Unveiling a Still-Undisclosed Biodiversity.

Autochthonous dairy lactic acid bacteria (LAB) isolates encompass a natural source of starter, adjunct, or probiotic candidates. In this context, traditionally manufactured, using exclusively animal rennet, Feta-type cheeses were collected from five farms located in different regions of Kefalonia island (Greece). The primary objective of this study was to isolate and characterize novel LAB, thereby exploring the unmapped microbial communities of Kefalonian Feta-type cheese and identifying new potential probiotics. The initial screening, included a preliminary gastrointestinal (GI) tolerance assessment (acidic conditions and bile salts), followed by their safety evaluation (hemolytic activity and antibiotic susceptibility). Based on the preliminary screening, selected strains underwent molecular identification and were further investigated for their probiotic attributes (lysozyme and phenol resistance, antimicrobial traits, antidiabetic aspects, cholesterol reduction and adhesion, adhesion to Caco-2 cells, and milk acidification potential). The results showed that 49, out of the 93 retrieved isolates, exhibited resistance to GI conditions, whereas 18 met the safety criteria. The molecular identification revealed strains belonging to the species Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and Lacticaseibacillus paracasei. The selected rod-shaped 14 isolates displayed a potential probiotic character. The best-performing isolates concerning cholesterol assimilation and adhesion, α-glucosidase inhibition, and epithelial adherence were Lpb. plantarum F89, F162, and F254 and Lcb. paracasei F214 and F216, whereas Lcb. paracasei F70 showed potential as a defined strain starter. The present study explores for the first time the biodiversity of traditionally fermented microbial communities in Kefalonian Feta-type cheese, revealing novel potential probiotic strains that can contribute to the development of innovative functional food products.

Spatiotemporal Dynamics of Assyrtiko Grape Microbiota.

Vitis vinifera, an economically significant grapevine species, is known for wine, juice, and table grape production. The berries of wine grapes host a diverse range of microorganisms influencing both grapevine health and the winemaking process. Indigenous to Greece, the emblematic variety Assyrtiko, renowned for high-quality white wines, originated from Santorini and spread to various Greek regions. Despite existing studies on the microbiota of several varieties, the carposphere microbiota of Assyrtiko grapes remains unexplored. Thus, we conducted a spatiotemporal metagenomic study to identify the epiphytic microbial community composition of Assyrtiko grapes. The study was conducted in two consecutive vintage years (2019 and 2020) across three different and distinct viticulture regions in Greece (Attica, Thessaloniki, Evros). We performed amplicon sequencing, targeting the 16S rRNA gene for bacteria and the ITS region for fungi, with subsequent comprehensive bioinformatic analysis. Our data indicate that the distribution and relative abundance of the epiphytic carposphere microbial communities of the Assyrtiko variety are shaped both by vintage and biogeography.

A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.

Effect of Free or Immobilized Lactiplantibacillus plantarum T571 on Feta-Type Cheese Microbiome.

BACKGROUND: Cheese microbiome plays a key role in determining the organoleptic and physico-chemical properties and may be also used as an authenticity tool for distinguishing probiotic cultures. Due to significant reduction of cell viability often witnessed during food production processes and storage, immobilization is proposed to ascertain high probiotic cell loads required to confer the potential health benefits. Hence, the aim of the present study was to investigate the effect of free or immobilized Lactiplantibacillus plantarum T571 on whey protein on feta cheese microbiome. METHODS: Next-Generation Sequencing technology was used to investigate cheese microbiome. Cheese samples containing free or immobilized Lactiplantibacillus plantarum T571 (a wild type strain isolated from Feta cheese brine) on whey protein, along with products containing commercial starter culture, were analyzed. RESULTS: The results showed a great diversity of bacteria and fungi genera among the samples. An increased presence of Lactobacillus OTUs in cheese with immobilized cells on whey protein was witnessed, highlighting the survival of the strain in the final product. The immobilized culture had also a significant impact on other genera, such as Lactococcus, Leuconostoc and Debaryomyces, which are associated with improved technological characteristics and health benefits. CONCLUSIONS: Enrichment of feta cheese with immobilized potential probiotics to secure cell viability consists of an industrial challenge and leads to distinct microbiome composition that may be used as a valuable food authenticity tool.

Genomic Insight Into Lacticaseibacillus paracasei SP5, Reveals Genes and Gene Clusters of Probiotic Interest and Biotechnological Potential.

The Lacticaseibacillus paracasei species is comprised by nomadic bacteria inhabiting a wide variety of ecological niches, from fermented foodstuffs to host-associated microenvironments. Lc. paracasei SP5 is a novel strain, originally isolated from kefir grains that presents desirable probiotic and biotechnological attributes. In this study, we applied genomic tools to further characterize the probiotic and biotechnological potential of the strain. Firstly, whole genome sequencing and assembly, were performed to construct the chromosome map of the strain and determine its genomic stability. Lc. paracasei SP5 carriers several insertion sequences, however, no plasmids or mobile elements were detected. Furthermore, phylogenomic and comparative genomic analyses were utilized to study the nomadic attributes of the strain, and more specifically, its metabolic capacity and ability to withstand environmental stresses imposed during food processing and passage through the gastrointestinal (GI) tract. More specifically, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-active enzyme (CAZymes) analyses provided evidence for the ability of the stain to utilize an array of carbohydrates as growth substrates. Consequently, genes for heat, cold, osmotic shock, acidic pH, and bile salt tolerance were annotated. Importantly bioinformatic analysis showed that the novel strain does not harbor acquired antimicrobial resistance genes nor virulence factors, in agreement with previous experimental data. Putative bacteriocin biosynthesis clusters were identified using BAGEL4, suggesting its potential antimicrobial activity. Concerning microbe-host interactions, adhesins, moonlighting proteins, exopolysaccharide (EPS) biosynthesis genes and pilins mediating the adhesive phenotype were, also, pinpointed in the genome of Lc. paracasei SP5. Validation of this phenotype was performed by employing a microbiological method and confocal microscopy. Conclusively, Lc. paracasei SP5 harbors genes necessary for the manifestation of the probiotic character and application in the food industry. Upcoming studies will focus on the mechanisms of action of the novel strain at multiple levels.

Whole-Genome Sequencing, Phylogenetic and Genomic Analysis of Lactiplantibacillus pentosus L33, a Potential Probiotic Strain Isolated From Fermented Sausages.

Lactobacillus is a diverse genus that includes species of industrial and biomedical interest. Lactiplantibacillus pentosus, formerly known as Lactobacillus pentosus, is a recently reclassified species, that contains strains isolated from diverse environmental niches, ranging from fermented products to mammalian gut microbiota. Importantly, several L. pentosus strains present health-promoting properties, such as immunomodulatory and antiproliferative activities, and are regarded as potential probiotic strains. In this study, we present the draft genome sequence of the potential probiotic strain L. pentosus L33, originally isolated from fermented sausages. Comprehensive bioinformatic analysis and whole-genome annotation were performed to highlight the genetic loci involved in host-microbe interactions and the probiotic phenotype. Consequently, we found that this strain codes for bile salt hydrolases, adhesins and moonlighting proteins, and for Class IIb bacteriocin peptides lacking the GxxxG and GxxxG-like motifs, crucial for their inhibitory activity. Its adhesion ability was also validated in vitro, on human cancer cells. Furthermore, L. pentosus L33 contains an exopolysaccharide (EPS) biosynthesis cluster, and it does not carry transferable antibiotic resistance genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and CAZymes analyses showed that L. pentosus L33 possesses biosynthetic pathways for seven amino acids, while it can degrade a wide array of carbohydrates. In parallel, Clusters of Orthologous Groups (COGs) and KEGG profiles of L. pentosus L33 are similar to those of 26 L. pentosus strains, as well as of two well documented L. plantarum probiotic strains. Conclusively, L. pentosus L33 exhibits good probiotic potential, although further studies are needed to elucidate the extent of its biological properties.

Genomic and Phylogenetic Analysis of Lactiplantibacillus plantarum L125, and Evaluation of Its Anti-Proliferative and Cytotoxic Activity in Cancer Cells.

Lactiplantibacillus plantarum is a diverse species that includes nomadic strains isolated from a variety of environmental niches. Several L. plantarum strains are being incorporated in fermented foodstuffs as starter cultures, while some of them have also been characterized as probiotics. In this study, we present the draft genome sequence of L. plantarum L125, a potential probiotic strain presenting biotechnological interest, originally isolated from a traditional fermented meat product. Phylogenetic and comparative genomic analysis with other potential probiotic L. plantarum strains were performed to determine its evolutionary relationships. Furthermore, we located genes involved in the probiotic phenotype by whole genome annotation. Indeed, genes coding for proteins mediating host-microbe interactions and bile salt, heat and cold stress tolerance were identified. Concerning the potential health-promoting attributes of the novel strain, we determined that L. plantarum L125 carries an incomplete plantaricin gene cluster, in agreement with previous in vitro findings, where no bacteriocin-like activity was detected. Moreover, we showed that cell-free culture supernatant (CFCS) of L. plantarum L125 exerts anti-proliferative, anti-clonogenic and anti-migration activity against the human colon adenocarcinoma cell line, HT-29. Conclusively, L. plantarum L125 presents desirable probiotic traits. Future studies will elucidate further its biological and health-related properties.