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Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), a culture based alternative for microbial diversity studies, is an attractive tool to dereplicate large numbers of isolates to a smaller set of 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 degrees C with 1 atm of overpressure. Proteolytic enzymes and alpha-amylase inactivated this curvaticin, while the effect of lipase was not severe.