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.

Microbial Profile Antibacterial Properties and Chemical Composition of Raw Donkey Milk.

The human interest in donkey milk is growing due to its nutritional, functional properties and excellent microbiological quality according to published reports. However, more research needs to be conducted to assess the above variables from various breeds. In the present study, milk samples were collected from 17 Cypriot and six Arcadian healthy Greek donkeys. The microbiological quality, somatic cell counts (SCC), chemical composition analysis, and antimicrobial activity of the samples was assessed. In addition, clustering and identification of the bacterial composition was performed by RAPD-PCR and 16S rDNA sequencing, respectively. The good microbiological quality of the samples as estimated by the total aerobic mesophilic and psychrotrophic counts, which ranged from 2.18 to 2.71 log CFU/mL and from 1.48 to 2.37 log CFU/mL, respectively, was also verified. SCC were below 4.4 log CFU/mL. However, potential pathogenic species of Staphylococcus aureus, Bacillus cereus, and Clostridium spp. were enumerated in the milk of both breeds. The gross chemical composition showed mean values for fat, protein, and lactose from 0.82% to 1.24%, 1.22% to 1.87%, and 6.01% to 6.78%, respectively. All milk samples exhibited an antimicrobial activity against St. haemolyticus and Listeria monocytogenes, although quality control measures should be taken for health and safety prior to human consumption.

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.

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.

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.

Evolution of microbial populations during traditional Feta cheese manufacture and ripening.

In three different dairies (A, B and C) located in Peloponess region (Southern Greece), traditional Feta cheese trials took place February to March using mixtures of sheep's and goat's milk. Only small variations in the evolution of microbial groups were observed during the whole ripening period. The main groups, such as thermophilic cocci, mesophilic lactococci, thermophilic lactobacilli, nonstarter lactic acid bacteria (NSLAB), presumptive Leuconostoc, enterococci and micrococci, reached their highest levels during the first 16 days, and then declined approximately 1-2 log units until the end of ripening. The remaining groups investigated, comprising yeasts, coliforms and Escherichia coli, were highest at day 4. The yeasts remained constant, while coliforms and E. coli decreased sharply and were not detectable after 120 days of ripening. A number of 146 isolates (dairy A) taken from all stages of the manufacturing and ripening process were purified and studied. Lactobacillus plantarum (58/146) and isolates of related species Lactobacillus pentosus and Lactobacillus paraplantarum (16/146) were the most common microorganisms found during cheese ripening. Streptococcus thermophilus (23/146) and Lactobacillus delbrueckii subsp. bulgaricus (20/146) were detected in high levels up to 20 days, and then gradually reduced. Enterococcus faecium (29/146) was found in all manufacturing and ripening stages.

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation.

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.