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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... more
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...
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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... more
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...
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Growth of and bacteriocin production by Streptococcus macedonicus ACA-DC 198 were assessed and modeled under conditions simulating Kasseri cheese production. Controlled fermentations were performed in milk supplemented with yeast extract... more
Growth of and bacteriocin production by Streptococcus macedonicus ACA-DC 198 were assessed and modeled under conditions simulating Kasseri cheese production. Controlled fermentations were performed in milk supplemented with yeast extract at different combinations of temperature (25, 40, and 55°C), constant pH (pHs 5 and 6), and added NaCl (at concentrations of 0, 2, and 4%, wt/vol). The data obtained were used to construct two types of predictive models, namely, a modeling approach based on the gamma concept, as well as a model based on artificial neural networks (ANNs). The latter computational methods were used on 36 control fermentations to quantify the complex relationships between the conditions applied (temperature, pH, and NaCl) and population behavior and to calculate the associated biokinetic parameters, i.e., maximum specific growth and cell count decrease rates and specific bacteriocin production. The functions obtained were able to estimate these biokinetic parameters fo...
Research Interests: Kinetics, Biology, Medicine, Multidisciplinary, Production economics, and 15 morePopulation, Bacteriocins, Animals, Applied Environmental Microbiology, Prediction Model, Bacteriocin, Milk, Cheese, Fermentation, Artificial Neural Network, Sodium Chloride, Biological Data, Computational Method, Specific Growth Rate, and Biological System
Abstract Staka is a naturally fermented cream produced from sheep or mixtures of sheep and goat milk in the island of Crete, Greece. This work is the first report on Staka cream microbiota. Initially, counts of various bacterial groups... more
Abstract Staka is a naturally fermented cream produced from sheep or mixtures of sheep and goat milk in the island of Crete, Greece. This work is the first report on Staka cream microbiota. Initially, counts of various bacterial groups were enumerated using selective media and growth conditions. All isolates (101) were subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis; the spectra obtained were compared with those of an in-house reference database. While 70 isolates were considered identified at the species level, the remaining 31 were not, probably due to a lack of, or an insufficient number of, reference spectra in the MALDI-TOF MS database. Strains representative of the observed diversity among the unidentified MALDI-TOF MS spectra were therefore subjected to pheS and 16S rRNA gene sequencing analysis for further identification. The identification results revealed the presence of Lactobacillus spp., Enterococcus spp., Streptococcus thermophilus and Aerococcus viridans.
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Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that... more
Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of...
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An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration... more
An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu 2+ , Hg 2+ , and Zn 2+ ). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro- p -nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin ...
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Kefir is a high nutritional fermented dairy beverage associated with a wide range of health benefits. It constitutes a unique symbiotic association, comprising mainly lactic acid bacteria, yeasts, and occasionally acetic acid bacteria,... more
Kefir is a high nutritional fermented dairy beverage associated with a wide range of health benefits. It constitutes a unique symbiotic association, comprising mainly lactic acid bacteria, yeasts, and occasionally acetic acid bacteria, which is strongly influenced by the geographical origin of the grains, the type of milk used, and the manufacture technology applied. Until recently, kefir microbiota has been almost exclusively studied by culture-dependent techniques. However, high-throughput sequencing, alongside omics approaches, has revolutionized the study of food microbial communities. In the present study, the bacterial, and yeast/fungal microbiota of four home-made samples (both grains and drinks), deriving from well spread geographical regions of Greece, and four industrial beverages, was elucidated by culture-dependent and -independent analyses. In all samples, classical microbiological analysis revealed varying populations of LAB and yeasts, ranging from 5.32 to 9.60 log CF...
Research Interests: Biology and Metagenomics
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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.... more
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 hea...
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Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), a culture based alternative for microbial diversity studies, is an attractive tool to dereplicate large numbers of isolates to a smaller set of... more
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), a culture based alternative for microbial diversity studies, is an attractive tool to dereplicate large numbers of isolates to a smaller set of representatives for downstream characterization. In the present study, MALDI-TOF MS, combined with a database of reference spectra compiled in previous studies, was applied to identify 88 non-starter lactic acid bacteria (NSLAB) isolated from 18 samples of four different artisanal cheeses produced in the Island of Naxos, Greece, from raw sheep and goat milk without the addition of starters. Eighty-four isolates (95.5%) could be identified directly via MALDI-TOF MS. Lactobacillus brevis and Lactobacillus plantarum were the dominant species, followed by Lactococcus lactis, Leuconostoc mesenteroides Lactobacillus paracasei, Lactobacillus rhamnosus, Pediococcus pentosaceus and Enterococcus faecium. The remaining four isolates represented species present in the database; however, within-species diversity was insufficiently covered. Additionally, pheS sequencing was applied to confirm identification.
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Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively... more
Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place ...
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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... more
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 14558(T) 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.
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Abstract 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... more
Abstract 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 16424T was confirmed by 16S rRNA gene sequence analysis, while propionic acid was first detected on day 7 and reached a concentration of 52.1 m m 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.
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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... more
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 ...
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ABSTRACT Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was... more
ABSTRACT Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, and reverse-phase chromatography. Bacteriocin is active at pH values between 4.0 and 9.0 and retains activity after incubation for 1h at 100°C. Proteolytic enzymes inactivated the bacteriocin after 1h of incubation, while renin resulted in full inactivation only after 24h. Lipase resulted in full inactivation after 4h. Applying molecular methods, it was determined that the bacteriocin produced, named as mesenterocin E131, was identical to mesenterocin Y105 and was expressed during the exponential growth phase.
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National Hellenic Research l'oundation, 4H Yassileos Constantinou Ave. Athens 116 35, (irecce Lipoxygenases (LOX) are enzymes that use molecular oxygen to produce hydroperoxides from fatty acids that have a cv.s.c/.v-pentadiene... more
National Hellenic Research l'oundation, 4H Yassileos Constantinou Ave. Athens 116 35, (irecce Lipoxygenases (LOX) are enzymes that use molecular oxygen to produce hydroperoxides from fatty acids that have a cv.s.c/.v-pentadiene structure A LOX form has been identified as an oil body membrane protein in cucumber and soybean cotyledons (1), and in anise seedlings (2) Oil bodies from olive seed endosperm and embryo tissues contain about 10% (w/w) protein and oleosins have been immunodetected (3). Recently we found that LOX activity is present in olive oil extracts (4). In this work LOX enzymatic activity has been detected in high molecular mass fractions of olive endosperm extracts and LOX is proposed to be an oil body membrane protein in olives Mature olives (Olea europaea cv microcarpa or mastoids) koroneiki variety, were generously gifted by the National Agricultural Research Foundation, Subtropics and Olive Institute of Chania, Greece Oil bodies of olive endosperms were isolate...
ABSTRACT Aminopeptidases from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were isolated. Enzymes were purified by chromatography on DEAE-cellulose and Sephadex G-150. The enzymes had molecular weights of... more
ABSTRACT Aminopeptidases from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were isolated. Enzymes were purified by chromatography on DEAE-cellulose and Sephadex G-150. The enzymes had molecular weights of 98,000 and 89,000 and optimal activity at pH 6.0 and 40°C and pH 6.5 and 35°C. respectively. The L. delbrueckii ssp. bulgaricus enzyme had higher activity on L-lysyl-4-nitroanilide than did the S. thermophilus enzyme. Both enzymes were inactivated by EDTA and 1,10-phenanthroline. Classical sulfhydryl and serine group reagents had little or no inhibitory effect on the enzymes; nevertheless, Cu++ and Hg++ resulted in strong inhibition; Ca++ stimulated the L. delbrueckii ssp. bulgaricus enzyme, and Mg++ stimulated the S. thermophilus enzyme.
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Abstract Samples of Greek virgin olive oils were examined for the presence of proteins and oxidative enzyme activities. All oil samples tested contained detectable amounts of protein, as well as lipoxygenase and polyphenol oxidase... more
Abstract Samples of Greek virgin olive oils were examined for the presence of proteins and oxidative enzyme activities. All oil samples tested contained detectable amounts of protein, as well as lipoxygenase and polyphenol oxidase activities. Sodium dodecyl sulfate-...
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The aim of the present work was the detection and enumeration of Streptococcus macedonicus in Greek traditional cheeses. A total of 68 traditional cheese samples have been examined. Enumeration was performed by plating on the S.... more
The aim of the present work was the detection and enumeration of Streptococcus macedonicus in Greek traditional cheeses. A total of 68 traditional cheese samples have been examined. Enumeration was performed by plating on the S. macedonicus differential ...
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Abstract: In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains... more
Abstract: In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-relatedInt. J. Mol. Sci. 2013, 14 4641
Research Interests: Microbiology, Evaluation, Industrial Biotechnology, Food Microbiology, Antibiotic Resistance, and 15 moreIn Vitro, Bacteriocins, Mice, Animals, Male, Bacteria, Microbial genetic and drug resistance, Animal health, DNA hybridization, Bacteriocin, Cheese, Gastrointestinal Tract, Microbial Sensitivity Tests, Food Sciences, and Bacterial translocation
Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of... more
Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of all microbial ...
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Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The... more
Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 degrees C with 1 atm of overpressure. Proteolytic enzymes and alpha-amylase inactivated this curvaticin, while the effect of lipase was not severe.
Research Interests: Microbiology, Kinetics, Medical Microbiology, Sequence Analysis, Food Microbiology, and 14 moreEnzymes, Bacteriocins, Listeria monocytogenes, Lactobacillus, Temperature, Fermentation, Meat products, Molecular weight, Amino Acid Sequence, Cation Exchange, Hydrogen-Ion Concentration, Ammonium Sulphate, Molecular Sequence Data, and Antonie van Leeuwenhoek
Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 ± 0.42 Da, as determined by electrospray mass spectrometry.... more
Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 ± 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558T, while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121°C with 1 at...
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In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%)... more
In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-related LAB was unexpected given their close phylogenetic relationship. Nevertheless, when tested in inhibition assays, the potency of the bacteriocin(s) of S. macedonicus ACA-DC 198 against the three oral streptoco...