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.

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.

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.

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.

Traditional low-alcoholic and non-alcoholic fermented beverages consumed in European countries: a neglected food group.

Fermented beverages hold a long tradition and contribution to the nutrition of many societies and cultures worldwide. Traditional fermentation has been empirically developed in ancient times as a process of raw food preservation and at the same time production of new foods with different sensorial characteristics, such as texture, flavour and aroma, as well as nutritional value. Low-alcoholic fermented beverages (LAFB) and non-alcoholic fermented beverages (NAFB) represent a subgroup of fermented beverages that have received rather little attention by consumers and scientists alike, especially with regard to their types and traditional uses in European societies. A literature review was undertaken and research articles, review papers and textbooks were searched in order to retrieve data regarding the dietary role, nutrient composition, health benefits and other relevant aspects of diverse ethnic LAFB and NAFB consumed by European populations. A variety of traditional LAFB and NAFB consumed in European regions, such as kefir, kvass, kombucha and hardaliye, are presented. Milk-based LAFB and NAFB are also available on the market, often characterised as 'functional' foods on the basis of their probiotic culture content. Future research should focus on elucidating the dietary role and nutritional value of traditional and 'functional' LAFB and NAFB, their potential health benefits and consumption trends in European countries. Such data will allow for LAFB and NAFB to be included in national food composition tables.

Survival, Intestinal Mucosa Adhesion, and Immunomodulatory Potential of Lactobacillus plantarum Strains.

Survival during transit through the gastrointestinal track, intestinal mucosa adhesion, and a potential immunomodulatory effect of Lactobacillus plantarum strains 2035 and ACA-DC 2640 were investigated in a rat model. According to microbiological and multiplex PCR analysis, both strains were detected in feces 24 h after either single-dose or daily administration for 7 days. Intestinal mucosa adhesion of L. plantarum 2035 was noted in the large intestine at 24 h after single-dose administration, while it was not detected at 48 h. Daily dosing, prolonged detection of the strain up to 48 h post-administration, and expanded adhesion to the small intestine. Adhesion of L. plantarum ACA-DC 2640 to the intestinal mucosa after single-dose administration was prolonged and more extended compared to L. plantarum 2035. Daily dosing increased both the levels and the rate of positive cultures of the strains compared to those of the single-dose scheme. In addition, both strains increased total IgG while decreased IgM and IgA serum levels. In conclusion, L. plantarum 2035 and L. plantarum ACA-DC 2640 survived transit through the gastrointestinal track, exhibited transient distinct adhesion to the intestinal mucosa and modulated the systemic immune response.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells.

Various Listeria monocytogenes strains may contaminate a single food product, potentially resulting in simultaneous exposure of consumers to multiple strains. However, due to bias in strain recovery, L. monocytogenes strains isolated from foods by selective enrichment (SE) might not always represent those that can better survive the immune system of a patient. We investigated the effect of cocultivation in tryptic soy broth with 0.6% yeast extract (TSB-Y) at 10°C for 8 days on (i) the detection of L. monocytogenes strains during SE with the ISO 11290-1:1996/Amd 1:2004 protocol and (ii) the in vitro virulence of strains toward the Caco-2 human colon epithelial cancer cell line following exposure to simulated gastric fluid (SGF; pH 2.0)-HCl (37°C). We determined whether the strains which were favored by SE would be effective competitors under the conditions of challenges related to gastrointestinal passage of the pathogen. Interstrain competition of L. monocytogenes in TSB-Y determined the relative population of each strain at the beginning of SE. This in turn impacted the outcome of SE (i.e., favoring survival of competitors with better fitness) and the levels exposed subsequently to SGF. However, strong growth competitors could be outcompeted after SGF exposure and infection of Caco-2 cells by strains outgrown in TSB-Y and underdetected (or even missed) during enrichment. Our data demonstrate a preferential selection of certain L. monocytogenes strains during enrichments, often not reflecting a selective advantage of strains during infection. These findings highlight a noteworthy scenario associated with the difficulty of matching the source of infection (food) with the L. monocytogenes isolate appearing to be the causative agent during listeriosis outbreak investigations.IMPORTANCE This report is relevant to understanding the processes involved in selection and prevalence of certain L. monocytogenes strains in different environments (i.e., foods or sites of humans exposed to the pathogen). It highlights the occurrence of multiple strains in the same food as an important aspect contributing to mismatches between clinical isolates and infection sources during listeriosis outbreak investigations.

Microemulsions as Potential Carriers of Nisin: Effect of Composition on Structure and Efficacy.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

Evaluation of Two Lactic Acid Bacteria Starter Cultures for the Fermentation of Natural Black Table Olives (Olea europaea L cv Kalamon).

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.

Comparative genomics of the dairy isolate Streptococcus macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus complex.

BACKGROUND: Within the genus Streptococcus, only Streptococcus thermophilus is used as a starter culture in food fermentations. Streptococcus macedonicus though, which belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC), is also frequently isolated from fermented foods mainly of dairy origin. Members of the SBSEC have been implicated in human endocarditis and colon cancer. Here we compare the genome sequence of the dairy isolate S. macedonicus ACA-DC 198 to the other SBSEC genomes in order to assess in silico its potential adaptation to milk and its pathogenicity status. RESULTS: Despite the fact that the SBSEC species were found tightly related based on whole genome phylogeny of streptococci, two distinct patterns of evolution were identified among them. Streptococcus macedonicus, Streptococcus infantarius CJ18 and Streptococcus pasteurianus ATCC 43144 seem to have undergone reductive evolution resulting in significantly diminished genome sizes and increased percentages of potential pseudogenes when compared to Streptococcus gallolyticus subsp. gallolyticus. In addition, the three species seem to have lost genes for catabolizing complex plant carbohydrates and for detoxifying toxic substances previously linked to the ability of S. gallolyticus to survive in the rumen. Analysis of the S. macedonicus genome revealed features that could support adaptation to milk, including an extra gene cluster for lactose and galactose metabolism, a proteolytic system for casein hydrolysis, auxotrophy for several vitamins, an increased ability to resist bacteriophages and horizontal gene transfer events with the dairy Lactococcus lactis and S. thermophilus as potential donors. In addition, S. macedonicus lacks several pathogenicity-related genes found in S. gallolyticus. For example, S. macedonicus has retained only one (i.e. the pil3) of the three pilus gene clusters which may mediate the binding of S. gallolyticus to the extracellular matrix. Unexpectedly, similar findings were obtained not only for the dairy S. infantarius CJ18, but also for the blood isolate S. pasteurianus ATCC 43144. CONCLUSIONS: Our whole genome analyses suggest traits of adaptation of S. macedonicus to the nutrient-rich dairy environment. During this process the bacterium gained genes presumably important for this new ecological niche. Finally, S. macedonicus carries a reduced number of putative SBSEC virulence factors, which suggests a diminished pathogenic potential.

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.

Macedovicin, the second food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198.

Streptococcus macedonicus ACA-DC 198 was found to produce a second lantibiotic named macedovicin in addition to macedocin. Macedovicin was purified to homogeneity and mass spectrometric analysis identified a peptide of approximately 3.4 kDa. Partial N-terminal sequence analysis and tandem mass spectrometry revealed that macedovicin was identical to bovicin HJ50 and thermophilin 1277 produced by Streptococcus bovis and Streptococcus thermophilus, respectively. Macedovicin inhibits a broad spectrum of lactic acid bacteria, several food spoilage species (e.g. Clostridium spp.) and oral streptococci. We determined the complete biosynthetic gene cluster of macedovicin. Even though the gene clusters of macedovicin, thermophilin 1277 and bovicin HJ50 were almost identical at the nucleotide level, there were important differences in their predicted genes and proteins. Bovicin HJ50-like lantibiotics were also found to be encoded by Streptococcus suis strains SC84 and D12, Enterococcus columbae PLCH2, Clostridium perfringens JGS1721 and several Bacillus strains. All these lantibiotics contained a number of conserved amino acids that may be important for their biosynthesis and activity, while phylogenetic analysis supported their dispersion by horizontal gene transfer. In conclusion, the production of multiple bacteriocins may enhance the bio-protective potential of S. macedonicus during food fermentation.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests.

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited ß-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Incidence of Bacteriocins Produced by Food-Related Lactic Acid Bacteria Active towards Oral Pathogens.

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 streptococci was high. Fourier-transform infrared spectroscopy combined with principal component analysis revealed that exposure of the target cells to the antimicrobial compounds caused major alterations of key cellular constituents. Our findings indicate that bacteriocins produced by food-related LAB against oral LAB may be rare, but deserve further investigation since, when discovered, they can be effective antimicrobials.

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.

Complete genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198.

The species Streptococcus macedonicus is associated with the food environment, especially with fermented dairy products. Here we present the complete 2.1-Mb genome sequence of strain ACA-DC 198, which was isolated from naturally fermented Greek kasseri cheese.

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.

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.

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.

In silico evidence for the horizontal transfer of gsiB, a σ(B)-regulated gene in gram-positive bacteria, to lactic acid bacteria.

gsiB, coding for glucose starvation-inducible protein B, is a characteristic member of the σ(Β) stress regulon of Bacillus subtilis and several other Gram-positive bacteria. Here we provide in silico evidence for the horizontal transfer of gsiB in lactic acid bacteria that are devoid of the σ(Β) factor.