The Lesvos sheep breed is an autochthonous Greek breed well adapted to the xerothermic conditions of Lesvos Island and is mainly exploited for milk production. In this study, 119 bacterial isolates from eight geographically well-spread samples of raw Lesvos breed sheep milk were identified. Based on rep-PCR genotypic profiling, 47 lactic acid bacteria (LAB) isolates were selected and evaluated for probiotic potential, with several among them exhibiting one or more probiotic traits. Notably, Limosilactobacillus fermentum 503 showed angiotensinconverting enzyme inhibitory (ACE-I), antimicrobial and gamma-aminobutyric acid (GABA) producing activities. Among Lacticaseibacillus casei/paracasei/rhamnosus isolates, strains 103 and 708 displayed ACE-I activity and adhered to collagen-coated plates, while strains 702 and 712 exhibited ACE-I, antimicrobial and bile salt hydrolase (BSH) activities. Streptococcus macedonicus 311 demonstrated ACE-I, antimicrobial and adhesion properties. Overall, raw milk from Lesvos breed sheep represents a rich and largely unexplored source of promising probiotic candidates for functional food development.

Donkey milk has gained increasing attention in recent years for its unique nutritional, medicinal, and cosmetic properties. This study employed a metataxonomic approach to investigate the microbiota of donkey milk samples collected from all farms operating in Greece during 2020-2021. Complementary chemical and culture-dependent microbiological analyses were performed alongside evaluation of milk fatty acid composition and antioxidant activity. Milk composition analyses showed no significant differences with published values, with variation observed only in fat content. Metataxonomic analysis identified the genera Acinetobacter, Enterococcus, Pseudomonas and Staphylococcus among the most abundant bacterial taxa, while Alternaria, Debaryomyces and Udeniomyces were the predominant yeast genera. Both classical microbiological and metataxonomic analysis confirmed the presence of lactic acid bacteria species. Several bacterial and fungal taxa correlated positively with donkey milk fatty acid content, while others negatively with milk total antioxidant capacity, thus affecting its composition and antioxidant potential.

Agiorgitiko is a major Greek red winegrape variety of high economic importance cultivated almost exclusively in the Nemea PDO zone in Peloponnese, Greece. Here, we describe the microbiota and mycobiota of grape and soil samples collected over three consecutive harvest periods (September 2019, 2020 and 2021). The results revealed a common microbiome composition across the Nemea PDO zone vineyards, despite significant variations in the community structure regarding dominant bacterial and fungal taxa per sampling year, which were associated with weather factors. Grape samples of 2019 were enriched in several plant growth promoting bacteria, including Bradyrhizobium, Streptomyces, Massilia and Sphingomonas, selected by the particular weather conditions. On the other hand, the predominance of Botrytis in the same samples was observed, indicating again the impact of weather conditions on the microbial structure. Understanding these dynamics could improve management practices aimed at vine cultivation and wine quality.

The comprehensive analysis of microbial communities reveals the unique microbial identity of different olive varieties, paving the way for new strategies in their development and commercial exploitation. In this context, the present study aimed to explore the microbial diversity and functional characteristics of Tsounati variety olives from the Monemvasia region of Peloponnese, Greece, that were naturally fermented for three months. The bacterial and fungal microbiota of both olives and brines were fingerprinted throughout the fermentation through classical microbiological analysis combined with molecular techniques. Among the 148 isolated bacteria, 85 were lactic acid bacteria (LAB), and 63 belonged to the Enterobacteriaceae family, while the 178 fungal isolates comprised 136 yeasts and 42 non-yeast or yeast-like fungi. Metataxonomic analysis confirmed the dominance of the bacterial genera Lactiplantibacillus, Leuconostoc, along with the Enterobacteriaceae family, and it revealed the presence of Coleofasciculaceae cyanobacteria mostly in olives. The dominant fungal genera were yeasts, namely Saccharomyces, Nakazawaea, and Cyberlindnera. Using the Folin–Ciocalteu assay, the average total polyphenol content of Tsounati fermented olive samples was 761.80 ± 128.87 mg gallic acid equivalents kg−1 after 90 days of fermentation. The concentrations of the triterpenic, maslinic, and oleanolic acids, as determined by HPLC, remained stable throughout fermentation, with average values of 4764 and 1807 mg kg−1, respectively. Finally, sensory analysis revealed the rich aromatic character of Tsounati variety, highlighting its potential to be used for Greek-style table olive production.

In the present work, we examined the angiotensin-I-converting enzyme-inhibitory (ACE-I) activity of the water soluble extracts of four long-ripened Feta cheese samples. High activity (>87 %) was determined for all of them. Additionally, we fingerprinted the non-starter lactic acid bacteria (NSLAB) isolated from the cheese samples. All NSLAB isolates were screened for their proteolytic activity and the most proteolytic ones were further evaluated for their ACE-I activity as well as ability to produce gamma-aminobutyric acid (GABA). The NSLAB isolates comprised 130 lactobacilli and 24 enterococci, with the dominant species being Levilactobacillus brevis, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei and Enterococcus faecium. Among them, 40 selected isolates exhibited high proteolytic activity, with 13 lactobacilli and two enterococci exhibiting strong ACE-I activity (>50 %), and six lactobacilli producing GABA. Thus, the hypertension risk associated with overconsumption of Feta cheese because of its high sodium content may be compensated by the anti-hypertensive traits of NSLAB microbiota members, which are active during the long cheese ripening time.

Staka is a traditional Greek sour cream made mostly from spontaneously fermented sheep milk or a mixture of sheep and goat milk. At the industrial scale, cream separators and starter cultures may also be used. Staka is sometimes cooked with flour to absorb most of the fat. In this study, we employed culture-based techniques, amplicon sequencing, and shotgun metagenomics to analyze the Staka microbiome for the first time. The samples were dominated by Lactococcus or Leuconostoc spp. Most other bacteria were lactic acid bacteria (LAB) from the Streptococcus and Enterococcus genera or Gram-negative bacteria from the Buttiauxella, Pseudomonas, Enterobacter, Escherichia-Shigella, and Hafnia genera. Debaryomyces, Kluyveromyces, or Alternaria were the most prevalent genera in the samples, followed by other yeasts and molds like Saccharomyces, Penicillium, Aspergillus, Stemphylium, Coniospotium, or Cladosporium spp. Shotgun metagenomics allowed the species-level identification of Lactococcus lactis, Lactococcus raffinolactis, Streptococcus thermophilus, Streptococcus gallolyticus, Escherichia coli, Hafnia alvei, Streptococcus parauberis, and Enterococcus durans. Binning of assembled shotgun reads followed by recruitment plot analysis of single reads could determine near-complete metagenome assembled genomes (MAGs). Culture-dependent and culture-independent analyses were in overall agreement with some distinct differences. For example, lactococci could not be isolated, presumably because they had entered a viable but not culturable (VBNC) state or because they were dead. Finally, several LAB, Hafnia paralvei, and Pseudomonas spp. isolates exhibited antimicrobial activities against oral or other pathogenic streptococci, and certain spoilage and pathogenic bacteria establishing their potential role in food bio-protection or new biomedical applications. Our study may pave the way for additional studies concerning artisanal sour creams to better understand the factors affecting their production and the quality.

Feta is the most renowned protected designation of origin (PDO) white brined cheese produced in Greece. The fine organoleptic characteristics and the quality of Feta rely on, among other factors, its overall microbial ecosystem. In this study, we employed 16S rDNA and internal transcribed spacer (ITS) amplicon sequencing, as well as shotgun metagenomics, to investigate the microbiome of artisanal homemade and industrial Feta cheese samples from different regions of Greece, which has very rarely been investigated. 16S rDNA data suggested the prevalence of the Lactococcus genus in the homemade samples, while Streptococcus and Lactobacillus genera prevailed in the industrial control samples. Species identification deriving from shotgun metagenomics corroborated these findings, as Lactococcus lactis dominated two homemade samples while Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were found to be dominating one industrial sample. ITS data revealed a complex diversity of the yeast population among the samples analyzed. Debaryomyces, Kluyveromyces, Cutaneotrichosporon, Pichia, Candida, and Rhodotorula were the major genera identified, which were distributed in a rather arbitrary manner among the different samples. Furthermore, a number of potential metagenome-assembled genomes (MAGs) could be detected among assembled shotgun bins. The overall analysis of the shotgun metagenomics supported the presence of different foodborne pathogens in homemade samples (e.g., Staphylococcus aureus, Listeria monocytogenes, Enterobacter cloacae, and Streptococcus suis), but with low to very low abundances. Concluding, the combination of both amplicon sequencing and shotgun metagenomics allowed us to obtain an in-depth profile of the artisanal homemade Feta cheese microbiome.

Cheese is characterized by a rich and complex microbiota that plays a vital role during both production and ripening, contributing significantly to the safety, quality, and sensory characteristics of the final product. In this context, it is vital to explore the microbiota composition and understand its dynamics and evolution during cheese manufacturing and ripening. Application of high-throughput DNA sequencing technologies have facilitated the more accurate identification of the cheese microbiome, detailed study of its potential functionality, and its contribution to the development of specific organoleptic properties. These technologies include amplicon sequencing, whole-metagenome shotgun sequencing, metatranscriptomics, and, most recently, metabolomics. In recent years, however, the application of multiple meta-omics approaches along with data integration analysis, which was enabled by advanced computational and bioinformatics tools, paved the way to better comprehension of the cheese ripening process, revealing significant associations between the cheese microbiota and metabolites, as well as their impact on cheese flavor and quality.

Limosilactobacillus fermentum ACA-DC 179, a strain with well documented probiotic properties, was included in Greek traditional Kaimaki ice cream produced using two different types of milk, namely plain and lactose-free cow milk. L fermentum survived throughout the ice cream manufacturing and storage. Viable counts did not decline during storage at -20 degrees C for 28 days both in plain (approximately 10(6) cfu g(-1)) and in lactose-free (approximately 10(8) cfu g(-1)) Kaimaki. Acidity and pH remained stable during storage, and, regarding fat and protein content, no variations were determined among samples belonging to the same type of ice cream. Melting characteristics of ice cream slightly altered due to strain addition. Ice cream containing L. fermentum was highly acceptable by the panellists evaluating sensory properties. To conclude, ice cream was proved to be a suitable matrix for L. fermentum ACA-DC 179 regarding the production of a probiotic functional food. (C) 2021 Elsevier Ltd. All rights reserved.

One of the main lactic acid bacterial species found in the kefir grain ecosystem worldwide is Lactobacillus kefiranofaciens, exhibiting strong auto-aggregation capacity and, therefore, being involved in the mechanism of grain formation. Its occurrence and dominance in kefir grains of various types of milk and geographical origins have been verified by culture-dependent and independent approaches using multiple growth media and regions of the 16S rRNA gene, respectively, highlighting the importance of their combination for its taxonomic identification. L. kefiranofaciens comprises two subspecies, namely kefiranofaciens and kefirgranum, but only the first one is responsible for the production of kefiran, the water-soluble polysaccharide, which is a basic component of the kefir grain and famous for its technological as well as health-promoting properties. L. kefiranofaciens, although very demanding concerning its growth conditions, can be involved in mechanisms affecting intestinal health, immunomodulation, control of blood lipid levels, hypertension, antimicrobial action, and protection against diabetes and tumors. These valuable bio-functional properties place it among the most exquisite candidates for probiotic use as a starter culture in the production of health-beneficial dairy foods, such as the kefir beverage.

Microbiological and physicochemical parameters of Lebanese Baladi goat milk from six farms were evaluated and 28 Lactobacillus strains were isolated and examined in vitro for their probiotic potential. Goat milk showed a balanced physicochemical composition while variable LAB counts were observed among samples. All isolates were identified as Lactobacillus rhamnosus. They exhibited diverse susceptibility to commonly used antibiotics, none was haemolytic and showed variable antimicrobial activity towards a range of spoilage and pathogenic bacteria. All strains remained unaffected at pH 3 and in the presence of bile salts (0.5%, w/v) for 3 h exhibiting also a partial bile salt hydrolase activity. Selected strains showed low adhesion to HT-29 and Caco-2 cells. Decrease in COX2 mRNA levels and a tendency to an increase in IL10 expression in human monocytes were noticed. This is the first report about probiotic and safety features of Lb. rhamnosus strains isolated from Lebanese Baladi goat milk. (c) 2021 Elsevier Ltd. All rights reserved.

Artisanal cheeses, in particular those prepared from raw milk, are the most commonly used ecosystems to mine microorganisms with both technological and human-health related potential. In the present study, the microbiota of two Greek artisanal cheeses, the PDO Kalathaki and the non-PDO Melichloro, was explored via classical microbiological analysis. Lactic acid bacteria isolated mainly belonged to the genus Enterococcus (66%) and to a lesser degree to Pediococcus (15%), Leuconostoc (8%), Lactobacillus (6%) and Lactococcus (5%). From the 112 initial isolates, 32 were selected and studied for their probiotic potential, i.e. safety traits, survival under gastrointestinal tract conditions, antimicrobial activity, adhesion, angiotensin I-converting enzyme inhibitory activity and immunomodulation of human monocytes. Two isolates, namely Lactobacillus brevis ACA-DC 1705 and Leuconostoc mesenteroides ACA-DC 1738, exhibited the most promising probiotic potential due to strong angiotensin I-converting enzyme inhibitory activity and anti-inflammatory modulation of immune cells, respectively, and, thus they could be used as adjuncts in novel functional products.

Bread is one of the most widely consumed foods in the world and has been an integral part of the human diet for thousands of years. Although yeasts are considered the major leavening agent in bread production, the use of sourdough as a natural starter in bread-making is one of the oldest food biotechnologies. Sourdough has been recently rediscovered and extensively studied as a cell factory able to modify cereals and other dough ingredients to functional and nutritional bread. This chapter provides a comprehensive overview about the sourdough microbiota, as this has been described so far by both conventional and omics approaches, as well as its functionality with regard to the technological properties of bread, such as texture, flavor, shelf-life, and nutritional value. Moreover, the emerging use of alternative raw materials for sourdough and bread production, the application of nonconventional starters, along with the adaptation of existing and the development of novel technologies in tailor-made bread types, as for example in the case of gluten-free bread, are also considered.

The characteristics of fermented milk are affected by the type of milk used and the microorganisms involved in the fermentation process. Goat milk has been widely suggested as a possible alternative to cow milk in allergic subjects, because of the high genetic variability in alpha-s1 casein (CSN1S1) content, which is associated with different technological and nutritional properties of milk. The aim of the study was to evaluate the suitability of goat milk with low and high CSN1S1 to produce fermented milk. In addition, the performance as starter of selected Lactobacillus paracasei FS109 strain compared to no-selected L. paracasei strains was investigated. Initially, the selected L. paracasei FS109 strain was tested for adhesion ability to HT-29 and Caco-2 cells and immunomodulation effect. Then, the strain was used to produce fermented milk from goat milk with a low and high casein CSN1S1 genotype. The results indicated that greater acidifying activity was obtained for L. paracasei FS109 after 24 h of fermentation than the other two strains tested independently by the CSN1S1 genotype. L. paracasei FS109 grew well during fermentation, reaching a higher value (>8.5 log CFU/mL). Interestingly, the same strain maintained a high viable population (about 9 log CFU/mL) during the 30-day cold storage of the product. The present study shows for the first time the suitability of the goat milk with low CSN1S1 genotypes to produce fermented milk and highlight the importance of strain selection in determination of technological and beneficial traits. Combining goat milk with low CSN1S1 and selected strains could be a strategy of improving traditional and functional fermented milk market.

The increased consumers' interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms. Triggered by the fact that artisanal food products can be a valuable source of novel probiotic strains, 106 lactic acid bacteria, all isolated from traditional Greek dairy products, namely Feta, Kasseri, Xynotyri, Graviera, Formaela, Galotyri, and Kefalotyri cheeses as well as yogurt and milk, were studied for probiotic properties. Based on their survival at pH 2.5 and their stability in the presence of bile salts, 20 strains were selected for further analysis. These strains exhibited diverse susceptibility to commonly used antibiotics, while none was hemolytic. Seven out of the 20 strains produced functional bile salt hydrolases in vitro. The only antimicrobial activity detected of Streptococcus thermophilus ACA-DC 26 against the oral pathogen Streptococcus mutans LMG 14558T was attributed to compound(s) of proteinaceous nature. Two Lactobacillus plantarum strains, namely ACA-DC 2640 and ACA-DC 4039, displayed the highest adhesion according to a collagen-based microplate assay and by using ΗΤ-29 and Caco-2 cells. Co-cultivation of THP-1 cells with selected strains indicated a tendency for anti-inflammatory modulation by Lactobacillus plantarum ACA-DC 2640 as well as Streptococcus thermophilus ACA-DC 26 and ACA-DC 170, as shown by an increase in IL10 mRNA levels. Moreover, milk cell-free supernatants of Lactobacillus plantarum ACA-DC 2640 and ACA-DC 4039 exhibited strong angiotensin I-converting enzyme inhibition. To conclude, new isolates presenting interesting probiotic features were described and should be further investigated as health-promoting factors.

Probiotic Feta cheese was produced using Propionibacterium freudenreichii subsp. shermanii as an adjunct since it has been shown to induce apoptosis of colon cancer cells in vitro and in vivo through the production of propionate and acetate. Microbiological and physicochemical analysis of the cheese was performed through the 60 d period of ripening. Counts of propionibacteria increased until day 7 and then remained constant until the end of ripening (approximately 9 log cfu g(-1)). Moreover, throughout ripening the presence of P. freudenreichii subsp. shermanii LMG 16424(T) was confirmed by 16S rRNA gene sequence analysis, while propionic acid was first detected on day 7 and reached a concentration of 52.1 mm on day 60. The ripened cheese containing P. freudenreichii subsp. shermanii was very well accepted by the sensory evaluation panellists. This is the first time that P. freudenreichii subsp. shermanii was studied as a probiotic adjunct in a white-brined cheese. (C) 2016 Elsevier Ltd. All rights reserved.

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of αS1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.

The production of Greek-style natural black table olives remains an empirical process relying on spontaneous fermentation despite its economic significance. For this reason producers often resort to increased NaCl concentration of the brine to secure quality of the product. In this study we employ two lactic acid bacteria Leuconostoc mesenteroides subsp. mesenteroides Lm139 and Lactobacillus pentosus DSM 16366 as starters in separate laboratory low salinity fermentations of "Kalamon" cultivar olives, processed according to the Greek-style method. L. mesenteroides subsp. mesenteroides Lm139 was previously isolated from Kalamon olives laboratory spontaneous fermentations, while L. pentosus DSM 16366 was isolated from fermenting green olives prepared according to the Spanish-style method. Spontaneous olives fermentation was also performed as a control. Microbiological and physicochemical analyses of the brines revealed that the use of the starters had a significant effect on the olives fermentation, leading to a faster acidification due to the more efficient consumption of soluble sugars in the brines. The final pH value reached by each starter culture used indicates a successful lactic fermentation. The production of lactic acid by the starters and the concomitant drop of the pH value proved to inhibit enterobacteria in a shorter period of time compared to the spontaneous fermentation. Concluding, the use of either of the two lactic acid bacteria as starters in Greek-style Kalamon olives fermentation could lead to a more controllable fermentation at lower salinities. The resulting product could be of higher quality with extended shelf-life while being at the same time safer for the consumer.

BACKGROUND:Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex.METHODOLOGY/PRINCIPAL FINDINGS:We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198's acquisition is not a recent event.CONCLUSIONS/SIGNIFICANCE:Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species.