Characterization of Bacterial Microbiota of P.D.O. Feta Cheese by 16S Metagenomic Analysis.

BACKGROUND: The identification of bacterial species in fermented PDO (protected designation of origin) cheese is important since they contribute significantly to the final organoleptic properties, the ripening process, the shelf life, the safety and the overall quality of cheese. METHODS: Ten commercial PDO feta cheeses from two geographic regions of Greece, Epirus and Thessaly, were analyzed by 16S metagenomic analysis. RESULTS: The biodiversity of all the tested feta cheese samples consisted of five phyla, 17 families, 38 genera and 59 bacterial species. The dominant phylum identified was Firmicutes (49% of the species), followed by Proteobacteria (39% of the species), Bacteroidetes (7% of the species), Actinobacteria (4% of the species) and Tenericutes (1% of the species). Streptococcaceae and Lactobacillaceae were the most abundant families, in which starter cultures of lactic acid bacteria (LAB) belonged, but also 21 nonstarter lactic acid bacteria (NSLAB) were identified. Both geographical areas showed a distinctive microbiota fingerprint, which was ultimately overlapped by the application of starter cultures. In the rare biosphere of the feta cheese, Zobellella taiwanensis and Vibrio diazotrophicus, two Gram-negative bacteria which were not previously reported in dairy samples, were identified. CONCLUSIONS: The application of high-throughput DNA sequencing may provide a detailed microbial profile of commercial feta cheese produced with pasteurized milk.

Sporopollenin Exine Microcapsules as Potential Intestinal Delivery System of Probiotics.

Despite several decades of research into encapsulation of bacteria, most of the proposed technologies are in the form of immobilized cultures. In this work, sporopollenin exine capsules (SECs) opened, using silica particles which act as pressing micro-probes, and loaded with Lactobacillus casei (L. casei) cells, are described for the first time. The proposed encapsulation provided ≈30× higher encapsulation yield (30.87%), compared to direct compression of SECs (0.99%). Encapsulated L. casei cells show 1.21- and 2.25-folds higher viability compared to free cells, in in vitro simulated fasted and fed media representing the human gastrointestinal (GI) tract, respectively. Encapsulated L. casei can proliferate inside the SECs, generating enough pressure to cause the SECs to burst and release the viable and metabolically active cells. The noticeable difference with the application of the SECs as a means of encapsulation is that the SECs may act as a bioreactor and provide time for the encapsulated cells to multiply thousands of times before being released, following the SEC's burst. The unique advantages of SECs alongside the proposed encapsulation method, demonstrates the potential application of SECs as delivery system of probiotics to the distal part of the human GI tract.

Magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis.

This paper evaluated magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis. Two different types of magnetic nanoparticles designated MPIO (iron concentration 2.5 mg/ml, size 1 µm) and NP (iron concentration 8.7 mg/ml, size 60 nm), both conjugated with S. aureus or S. enteritidis antibodies were evaluated as an enrichment procedure for PCR-detection of the pathogens in Trypticase Soy Broth, milk, blood and meat broth. Bacterial suspensions (1.5x108 cfu/ml) were prepared and serial diluted 10-1. The MPIO and NP nanoparticles were added, followed by incubation for 1 hour at room temperature, magnetic separation of the pellet, DNA extraction and PCR, targeting the femA and invA sequences. The nanoparticle-free and the NP-supplemented dilutions were positive down to the 1.5x102 cfu/ml concentration for both bacteria. The MPIO-supplemented dilutions were positive down to approx. 2x100 cfu/ml concentration, respectively. Bacteria-free TSB was negative by PCR. MPIO nanoparticles (size 1 µm) enhanced the detection of S. aureus and S. enteritidis by PCR, whilst NP nanoparticles (size 60 nm) did not, thus indicating that the size of the magnetic nanoparticles play a significant role in the enrichment procedure.

High level of heterogeneity among Listeria monocytogenes isolates from clinical and food origin specimens in Greece.

In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.

Improvement of oleuropein extractability by optimising steam blanching process as pre-treatment of olive leaf extraction via response surface methodology.

Impact of steam, hot water blanching and UV-C irradiation as pre-treatments on extraction of oleuropein and related biophenols from olive leaves (OLs), was investigated. Moreover, particle size effect of olive leaves and steam blanching duration were selected as independent variables to optimise steam blanching process in terms of oleuropein content (OC) and antioxidant activity (AC) of ethanolic extracts, by using response surface methodology. Optimum conditions for OC and AC were 10 min steam blanching of 20-11 and 3-1mm olive leaf fraction, respectively. Depending on the extraction procedure, at optimum conditions of steaming the results indicate that steam blanching of OL prior to extraction can significantly increase oleuropein yield from 25 to 35 times compared to non-steam blanched sample, whereas the antioxidant activity increased from 4 to 13 times. No significant UV-C effect was observed in OC and AC, while hot water blanched samples showed significantly higher oleuropein yields and antioxidant activity compared to untreated samples.

Encapsulation of complex extracts in beta-cyclodextrin: an application to propolis ethanolic extract.

Propolis ethanolic extracts (PE) are rather complicated mixtures of bioactive compounds belonging to several chemical classes. The potential use of beta-cyclodextrin (beta-CD) cavity for the incorporation of specific PE components, aiming to increase their solubility in water, was studied in a Greek propolis, which was rich in polyphenols and terpenes. The PE/beta-CD inclusion complexes were prepared by sonication of PE suspensions in aqueous solutions of beta-CD, followed by filtration and freeze-drying. The aqueous solubility of PE in the presence of beta-CD was studied by the construction of solubility diagrams and by determining the fraction of PE constituents that was dissolved in water. Encapsulation efficiencies were found to be higher (9.4-23.3%) for relatively small aromatic molecules like cinnamic and benzoic acid derivatives and lower for terpenic acids (5.0-6.7%), anthraquinones (3.6-8.4%) and flavonoids (4.0-10.7%). The respective in vitro solubilities in simulated gastric fluid followed an opposite trend, being lower for the relatively small aromatic molecules. It is concluded that the encapsulation in beta-CD may increase the solubility of PE constituents in a manner related to their structure, while the amount of substances released will depend both on their chemical properties and on their relative abundance in the matrix.

Effects of gamma radiation on microbiological status, fatty acid composition, and color of vacuum-packaged cold-stored fresh pork meat.

Pork meat samples were inoculated with high or low levels (10(6) or 10(3) CFU/g) of Salmonella Enteritidis, vacuum packaged, exposed to gamma radiation (1.0, 2.5, and 4.7 kGy), and stored for 1 month at 4 +/- 1 degrees C. In highly contaminated samples, the target strain was completely eliminated only by the 4.7 kGy radiation dose, whereas in samples at the lower contamination level, 2.5 kGy was sufficient to eliminate Salmonella Enteritidis. The highest of the applied radiation doses reduced the aerobic microflora and extended the sample's refrigeration shelf life by at least 2 weeks. The fatty acid profile of pork meat was not significantly affected by any of the applied radiation doses. Irradiation increased the proportion of saturated fatty acids (SFA) and decreased the content of the polyunsaturated fatty acids (P < 0.05). Irradiation also affected negatively the proportions of the nutritional indexes omega-6/omega-3, SFA/monounsaturated fatty acids, and SFA/polyunsaturated fatty acids. The proportion of the trans fatty acids C18:1omega-9 t9 and C18:2 t9,t12 in the total fatty acids was nearly doubled (90 and 86%, respectively) in the samples that had been exposed to 4.7 kGy. None of the applied radiation doses changed the lightness (L* value) of the meat, but redness (a*) and yellowness (b*) increased, particularly for the samples treated with 4.7 kGy.

Effects of gamma-irradiation on Listeria monocytogenes population, colour, texture and sensory properties of Feta cheese during cold storage.

Feta, a white brine cheese, was produced and contaminated with Listeria monocytogenes. Contamination occurred either at the beginning (pre-process contamination) or at the end of Feta manufacturing (post-process contamination). In the first case the milk was contaminated with 10(3) cfu/ml, and 2 months later, in the final product, the L. monocytogenes population was approximately 10(5) cfu/g. In the second case, the brine (NaCl, 7% w/v), in which the Feta was packaged, was contaminated with 10(3) cfu/ml. Contaminated Feta samples were vacuum-packaged and exposed to irradiation doses of 1.0, 2.5 and 4.7 kGy and stored at 4 degrees C for a month. In the pre-process contaminated samples none of the irradiation doses eliminated L. monocytogenes; however the highest dose reduced the viable population to a level which is in compliance with EC regulations. In the post-process contamination, the 2.5 kGy and 4.7 kGy doses reduced L. monocytogenes counts below the detection limit. Irradiation had no effect on the texture of Feta. Irradiation at 4.7 kGy increased Feta's redness and decreased its yellowness and lightness. Sensorial analyses showed that at the 4.7 kGy dose, the aroma profile of Feta was temporarily affected, since it was restored after 30 days of cold storage.

Microbial population, physicochemical quality, and allergenicity of molluscs and shrimp treated with cobalt-60 gamma radiation.

Frozen molluscs (squid, octopuses, and cuttlefish) and crustaceans (shrimp) were irradiated using a cobalt-60 gamma source, at different doses, in order to investigate the effects of gamma radiation on their microbial population, organoleptic characteristics, lipid profile, and tropomyosin content. Irradiation of shrimp and squid with either 2.5 or 4.7 kGy reduced mesophilic bacteria contamination to low or nondetectable levels, respectively, whereas irradiation of octopus and cuttlefish with the same doses reduced the bacterial population. Irradiation treatment had no significant (P > 0.05) effect on the total lipid content and the major detected classes of polar and neutral lipids, whereas it significantly (P < 0.05) increased the contents of neutral lipids in octopus mantle and in shrimp muscle and cephalothorax samples. The total fatty acid content and the omega-3: omega-6 fatty acid ration was not affected. A dose-dependent significant (P < 0.05) decrease in the ratio of polyunsaturated fatty acids:saturated fatty acids was observed. With the increase in radiation dose, redness (a) and yellowness (b) values showed a variation, whereas the lightness (L) value was significantly (P < 0.05) decreased in mollusc mantles and shrimp muscle and increased in shrimp cephalothorax. The total of color changes ( delta E) increased (P < 0.05) as the dose increased. Significant (P < 0.05) changes in textural properties were observed with radiation treatment in octopus tentacles and in squid and cuttlefish mantle. The amount of tropomyosin, which is the major mollusc and crustacean allergen in the irradiated organisms, was reduced by gamma radiation, depending on the dose.