Study of the Microbiome of the Cretan Sour Cream Staka Using Amplicon Sequencing and Shotgun Metagenomics and Isolation of Novel Strains with an Important Antimicrobial Potential.

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.

Screening of New Industrially Important Bacterial Strains for 1,3-Propanediol, 2,3-Butanediol and Ethanol Production through Biodiesel-Derived Glycerol Fermentations.

A study on the ability of new microbial strains to assimilate biodiesel-derived glycerol at low purity (75% w/w) and produce extra-cellular platform chemical compounds of major interest was carried out. After screening several bacterial strains under different fermentation conditions (e.g., pH, O2 availability, glycerol purity), three of the screened strains stood out for their high potential to produce valued-added products such as 2,3-butanediol (BDO), 1,3-propanediol (PDO) and ethanol (EtOH). The results indicate that under aerobic conditions, Klebsiella oxytoca ACA-DC 1581 produced BDO in high yield (YBDO/Gly = 0.46 g/g, corresponding to 94% of the maximum theoretical yield; Ymt) and titer, while under anaerobic conditions, Citrobacter freundii NRRL-B 2645 and Enterobacter ludwigii FMCC-204 produced PDO (YPDO/Gly = 0.56 g/g, 93% of Ymt) and EtOH (YEtOH/Gly = 0.44 g/g, 88% of Ymt), respectively. In the case of C. freundii, the regulation of pH proved to be mandatory, due to lactic acid production and a subsequent drop of pH that resulted in fermentation ceasing. In the fed-batch culture of K. oxytoca, the BDO maximum titer reached almost 70 g/L, the YBDO/Gly and the mean productivity value (PrBDO) were 0.47 g/g and 0.4 g/L/h, respectively, while no optimization was imposed. The final BDO production obtained by this wild strain (K. oxytoca) is among the highest in the international literature, although the bioprocess requires optimization in terms of productivity and total cost. In addition, for the first time in the literature, a strain from the species Hafnia alvei (viz., Hafnia alvei ACA-DC 1196) was reported as a potential BDO producer. The strains as well as the methodology proposed in this study can contribute to the development of a biorefinery that complements the manufacture of biofuels with high-value biobased chemicals.

Short-term effects of goat milk yogurt-containing angiotensin-converting enzyme inhibitory peptides and two raisin varieties on subjective appetite, blood pressure and glycaemic responses in healthy adults. Results from a randomised clinical trial.

Goat milk yogurt (GMY) and raisins are popular foods with a favourable nutrient profile. Our aim was to determine the glycaemic index (GI) and postprandial responses to GMY-containing angiotensin-converting enzyme inhibitory (ACE-I) peptides carrying the RPKHPINHQ isracidin fragment and two Greek raisin varieties in an acute feeding setting. A total of twelve healthy participants (four male and eight female) consumed breakfast study foods containing 25 g available carbohydrate on seven occasions over a 3- to 9-week period: food 1: D-glucose (25 g) served as the control and was consumed on three separate occasions; food 2: GMY (617·28 g); food 3: Corinthian raisins (37·76 g); food 4: Sultana raisins (37·48 g) and food 5: GMY & C (308·64 g GMY and 18·88 g C). Postprandial glucose was measured over a 2 h period for the determination of GI and glycaemic load (GL). Subjective appetite ratings (hunger, fullness and desire to eat) were assessed by visual analogue scales (100 mm) at 0­120 min. Blood pressure (systolic and diastolic; BP) was measured at baseline and 120 min. GMY provided low GI (26), C and S provided high GI/low GL (75/10 and 70/9, respectively) and GMYC provided low GI (47) values on glucose scale compared with D-glucose. Peak blood glucose rise was significantly lower only for GMY and GMYC compared with reference food (D-Glucose), as well as C and S (Pfor all < 0·05). No differences were observed between test foods for fasting glucose, BP and subjective appetite. In conclusion, GMY and GMYC attenuated postprandial glycaemic responses, which may offer advantages to glycaemic control.

Comparison of the Microbiome of Artisanal Homemade and Industrial Feta Cheese through Amplicon Sequencing and Shotgun Metagenomics.

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.

Omics Approaches to Assess Flavor Development in Cheese.

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.

In Vitro Virulence Potential, Surface Attachment, and Transcriptional Response of Sublethally Injured Listeria monocytogenes following Exposure to Peracetic Acid.

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes (motB and flaA) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA. Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

The Impact of Different Inoculation Schemes on the Microbiota, Physicochemical and Sensory Characteristics of Greek Kopanisti Cheese throughout Production and Ripening.

Kopanisti is a Greek PDO cheese, which is traditionally produced by the addition of an amount of over-mature Kopanisti, called Mana Kopanisti, to initiate cheese ripening. The aim of this study was the production of four types of Kopanisti cheese (A-D) using pasteurized cow milk, and a combination of the following starters/adjuncts in order to test their ability to be used in Kopanisti cheese production: A: Lactococcus lactis subsp. lactis and Lacticaseibacillus paracasei, B: L. lactis and Lc. paracasei/Mana Kopanisti, C: L. lactis and Lc. paracasei/Ligilactobacillus acidipiscis and Loigolactobacillus rennini, D: Lig. acidipiscis and Loig. rennini. Throughout production and ripening, classical microbiological, metataxonomics and physicochemical analyses were employed, while the final products (Day 35) were subjected to sensory analysis as well. Most interestingly, beta-diversity analysis of the metataxonomics data revealed the clusters constructed among the Kopanisti types based on the different inoculation schemes. On day 35, Kopanisti A-C types clustered together due to their similar 16S microbiota, while Kopanisti D was highly differentiated. On the contrary, ITS data clustered Kopanisti B and C together, while Kopanisti A and D were grouped seperately. Finally, based on the sensory evaluation, Kopanisti C appeared to have the most suitable bacteria cocktail for the Kopanisti cheese production. Therefore, not only were the conventional starters used, but also the Lig. acidipiscis and Loig. rennini strains could be used in a standardized Kopanisti cheese production that could lead to final products of high quality and safety.

Lactobacillus kefiranofaciens: From Isolation and Taxonomy to Probiotic Properties and Applications.

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.

Zooming Into the Microbiota of Home-Made and Industrial Kefir Produced in Greece Using Classical Microbiological and Amplicon-Based Metagenomics Analyses.

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 CFU mL-1 or g-1, and 2.49 to 7.80 log CFU mL-1 or g-1, respectively, while in two industrial samples no yeasts were detected. Listeria monocytogenes, Salmonella spp. and Staphylococcus spp. were absent from all the samples analyzed, whereas Enterobacteriaceae were detected in one of them. From a total of 123 isolates, including 91 bacteria and 32 yeasts, Lentilactobacillus kefiri, Leuconostoc mesenteroides, and Lactococcus lactis as well as Kluvyeromyces marxianus and Saccharomyces cerevisiae were the mostly identified bacterial and yeast species, respectively, in the home-made samples. On the contrary, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lacticaseibacillus rhamnosus along with Debaryomyces hansenii and K. marxianus were the main bacterial and yeast species, respectively, isolated from the industrial beverages. In agreement with the identification results obtained from the culture-dependent approaches, amplicon-based metagenomics analysis revealed that the most abundant bacterial genera in almost all home-made samples (both grains and drinks) were Lactobacillus and Lactococcus, while Saccharomyces, Kazachstania, and Kluvyeromyces were the predominant yeasts/fungi. On the other hand, Streptococcus, Lactobacillus, and Lactococcus as well as Kluvyeromyces and Debaryomyces dominated the bacterial and yeast/fungal microbiota, respectively, in the industrial beverages. This is the first report on the microbiota of kefir produced in Greece by a holistic approach combining classical microbiological, molecular, and amplicon-based metagenomics analyses.

Isotopic Traceability (13C and 18O) of Greek Olive Oil.

In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the 18O and 13C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ18O isotopic values range from 19.2 ‱ to 25.2 ‱ and the δ13C values range from -32.7 ‱ to -28.3 ‱. These differences between the olive oils' isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the 13C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.

Differential Modulation of Listeria monocytogenes Fitness, In Vitro Virulence, and Transcription of Virulence-Associated Genes in Response to the Presence of Different Microorganisms.

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCEListeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.

Unraveling the Microbiota of Natural Black cv. Kalamata Fermented Olives through 16S and ITS Metataxonomic Analysis.

Kalamata natural black olives are one of the most economically important Greek varieties. The microbial ecology of table olives is highly influenced by the co-existence of bacteria and yeasts/fungi, as well as the physicochemical parameters throughout the fermentation. Therefore, the aim of this study was the identification of bacterial and yeast/fungal microbiota of both olives and brines obtained from 29 cv. Kalamata olive samples industrially fermented in the two main producing geographical regions of Greece, namely Aitoloakarnania and Messinia/Lakonia. The potential microbial biogeography association between certain taxa and geographical area was also assessed. The dominant bacterial family identified in olive and brine samples from both regions was Lactobacillaceae, presenting, however, higher average abundances in the samples from Aitoloakarnania compared to Messinia/Lakonia. At the genus level, Lactobacillus, Celerinatantimonas, Propionibacterium and Pseudomonas were the most abundant. In addition, the yeasts/fungal communities were less diverse compared to those of bacteria, with Pichiaceae being the dominant family and Pichia, Ogataea, and Saccharomyces being the most abundant genera. To the best of our knowledge, this is the first report on the microbiota of both olives and brines of cv. Kalamata black olives fermented on an industrial scale between two geographical regions of Greece using metagenomics analysis.

MALDI-TOF MS profiling of non-starter lactic acid bacteria from artisanal cheeses of the Greek island of Naxos.

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.

Whole-genome sequence data and analysis of Lactobacillus delbrueckii subsp. lactis ACA-DC 178 isolated from Greek Kasseri cheese.

Lactobacillus delbrueckii subsp. lactis is employed in the production of various types of cheese. Here, we report the complete genome sequence of L. lactis ACA-DC 178 isolated from Greek Kasseri cheese. The chromosome of ACA-DC 178 contains 2,050,316 bp with a GC content of 49.6%. A total of 2,112 genes were identified in the genome sequence including 1,752 protein-coding genes, 239 putative pseudogenes, 94 tRNA and 27 rRNA genes. According to the COG annotation, about 80% of the protein-coding genes (1,417 proteins) were assigned to at least one functional category. Approximately the 1/3 of these proteins were distributed among three categories, namely replication, recombination and repair (category L: 10.6%), translation, ribosomal structure and biogenesis (category J: 7.5%) and amino acid transport and metabolism (category E: 7.2%). Fourteen integrated GIs with a total of 159 genes were found in ACA-DC 178 genome. Several of these genes encode proteins associated with exopolysaccharide biosynthesis, amino acid transport and subunits of restriction-modification systems. One large CRISPR array of 3,197 bp containing 52 spacers, several of which are identical to phage sequences having hosts in the genus Lactobacillus, was also identified. The annotated genome sequence of L. lactis ACA-DC 178 is deposited at the European Nucleotide Archive under the accession number LS991409. Raw reads are deposited in the Sequence Read Archive (SRR8866601-3).

Comparative Genomics of Streptococcus thermophilus Support Important Traits Concerning the Evolution, Biology and Technological Properties of the Species.

Streptococcus thermophilus is a major starter for the dairy industry with great economic importance. In this study we analyzed 23 fully sequenced genomes of S. thermophilus to highlight novel aspects of the evolution, biology and technological properties of this species. Pan/core genome analysis revealed that the species has an important number of conserved genes and that the pan genome is probably going to be closed soon. According to whole genome phylogeny and average nucleotide identity (ANI) analysis, most S. thermophilus strains were grouped in two major clusters (i.e., clusters A and B). More specifically, cluster A includes strains with chromosomes above 1.83 Mbp, while cluster B includes chromosomes below this threshold. This observation suggests that strains belonging to the two clusters may be differentiated by gene gain or gene loss events. Furthermore, certain strains of cluster A could be further subdivided in subgroups, i.e., subgroup I (ASCC 1275, DGCC 7710, KLDS SM, MN-BM-A02, and ND07), II (MN-BM-A01 and MN-ZLW-002), III (LMD-9 and SMQ-301), and IV (APC151 and ND03). In cluster B certain strains formed one distinct subgroup, i.e., subgroup I (CNRZ1066, CS8, EPS, and S9). Clusters and subgroups observed for S. thermophilus indicate the existence of lineages within the species, an observation which was further supported to a variable degree by the distribution and/or the architecture of several genomic traits. These would include exopolysaccharide (EPS) gene clusters, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-CRISPR associated (Cas) systems, as well as restriction-modification (R-M) systems and genomic islands (GIs). Of note, the histidine biosynthetic cluster was found present in all cluster A strains (plus strain NCTC12958T) but was absent from all strains in cluster B. Other loci related to lactose/galactose catabolism and urea metabolism, aminopeptidases, the majority of amino acid and peptide transporters, as well as amino acid biosynthetic pathways were found to be conserved in all strains suggesting their central role for the species. Our study highlights the necessity of sequencing and analyzing more S. thermophilus complete genomes to further elucidate important aspects of strain diversity within this starter culture that may be related to its application in the dairy industry.

Reverse micelles as nano-carriers of nisin against foodborne pathogens. Part II: The case of essential oils.

During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure.

Comparative Genomics of Lactobacillus acidipiscis ACA-DC 1533 Isolated From Traditional Greek Kopanisti Cheese Against Species Within the Lactobacillus salivarius Clade.

Lactobacillus acidipiscis belongs to the Lactobacillus salivarius clade and it is found in a variety of fermented foods. Strain ACA-DC 1533 was isolated from traditional Greek Kopanisti cheese and among the available L. acidipiscis genomes it is the only one with a fully sequenced chromosome. L. acidipiscis strains exhibited a high degree of conservation at the genome level. Investigation of the distribution of prophages and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) among the three strains suggests the potential existence of lineages within the species. Based on the presence/absence patterns of these genomic traits, strain ACA-DC 1533 seems to be more related to strain JCM 10692T than strain KCTC 13900. Interestingly, strains ACA-DC 1533 and JCM 10692T which lack CRISPRs, carry two similar prophages. In contrast, strain KCTC 13900 seems to have acquired immunity to these prophages according to the sequences of spacers in its CRISPRs. Nonetheless, strain KCTC 13900 has a prophage that is absent from strains ACA-DC 1533 and JCM 10692T. Furthermore, comparative genomic analysis was performed among L. acidipiscis ACA-DC 1533, L. salivarius UCC118 and Lactobacillus ruminis ATCC 27782. The chromosomes of the three species lack long-range synteny. Important differences were also determined in the number of glycobiome related proteins, proteolytic enzymes, transporters, insertion sequences and regulatory proteins. Moreover, no obvious genomic traits supporting a probiotic potential of L. acidipiscis ACA-DC 1533 were detected when compared to the probiotic L. salivarius UCC118. However, the existence of more than one glycine-betaine transporter within the genome of ACA-DC 1533 may explain the ability of L. acidipiscis to grow in fermented foods containing high salt concentrations. Finally, in silico analysis of the L. acidipiscis ACA-DC 1533 genome revealed pathways that could underpin the production of major volatile compounds during the catabolism of amino acids that may contribute to the typical piquant flavors of Kopanisti cheese.

Evaluating the probiotic potential and technological characteristics of yeasts implicated in cv. Kalamata natural black olive fermentation.

In the present study, 49 yeast strains previously isolated from cv. Kalamata table olive fermentation were assessed for their probiotic potential and technological characteristics. The probiotic assays included the in vitro survival in simulated gastric and pancreatic digestions, surface adhesion to the intestinal cell line Caco-2, hydrophobicity, autoaggregation and haemolytic activity. The technological features of the strains were also elucidated in terms of enzymatic activity and susceptibility to diverse salt levels (0-250 g/L) and pH values (3.5, 5.0, and 6.5). The obtained results indicated that during the simulated gastric and pancreatic digestions, 42 out of the 49 yeast strains presented overall survival rate higher than 50%, while 24 strains showed survival percentage higher than 70% at the end of the digestions. Furthermore, the majority of the assayed strains presented hydrophobicity percentage higher than 75%, while the autoaggregation ability ranged between 72 and 91%. None of the strains showed haemolytic activity. The majority of the strains presented high tolerance to salt with some strains exhibiting tolerance even at salt concentrations higher than 200 g/L. Concerning the enzymatic activity, 45 strains presented valine and cystine arylamidase activity, while positive reactions for the enzymes ß- and α-glucosidase were observed for 27 and 14 strains, respectively. Moreover, 11 strains presented α-galactosidase and alkaline phosphatase activity. From the total number of studied yeasts, the strain Y34 belonging to Saccharomyces cerevisiae presented positive results in the majority of both probiotic and technological assays and thus it could be considered a potential starter either as single or as combined culture with lactic acid bacteria in the fermentation of Greek-style natural black olives.

Reverse micelles as nanocarriers of nisin against foodborne pathogens.

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 when RMs are applied in different food matrices.

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions.

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.