A comparison for acid production, proteolysis, autolysis and inhibitory properties of lactic acid bacteria from fresh and mature Feta PDO Greek cheese, made at three different mountainous areas.

Isolates of NSLAB were obtained from fresh (58 isolates) and mature (38) Feta cheese made at household level in three different mountainous areas, in order to study the effect of the area of production on NSLAB composition and their technological characteristics. Results obtained by SDS-PAGE of whole-cell proteins indicated that the microflora of the fresh cheese was composed of either lactococci (areas 1, 2), or lactococci and enterococci (area 3). The NSLAB microflora of mature cheese was composed almost entirely of lactobacilli species, differing according to the area of production. Species allocation by the SDS-PAGE method was confirmed by sequencing representative strains. Lactococci of cheese made in area 1 exhibited a narrow spectrum of antibacterial activity compared to isolates from areas 2 and 3, while for lactobacilli from all three areas a similar spectrum was noticed. Lactococci from area 2 exhibited higher (P<0.05) mean acidifying activity than lactococci from area 1. The isolates from the three areas also differed in respect of their caseinolytic activity, with preferences towards ß-CN (areas 1 and 2) or αs-CN (area 3). Mean proteolytic activity of lactococci from area 1 was stronger (P<0.05) than that of lactococci from area 2 and the same was observed for their mean aminopeptidase activity, as well as their extent of autolysis at pH5.1. Mean acidifying activity of lactobacilli after 6h was for strains of area 3>2=1. The strains from areas 1 and 3 degraded preferentially αs-CN, while a clear preference towards ß-CN was noticed for strains of area 2; their mean proteolytic activity was for strains of area 1 higher (P<0.05) than strains from area 3. The above results suggest that cheeses from the three areas differ in species composition of NSLAB and their technological properties. Principal component analysis of results on acidifying and proteolytic activities as well as autolysis allowed the distinction of lactococci according to their derivation area enabling the selection of appropriate strains as starters for cheese production in each area.

The Microfloras of Traditional Greek Cheeses.

Many traditional cheeses are made in Greece. Some of them are, in fact, types of the same cheese variety, whether or not they have different cheesemaking technologies, but are known by different local names. Twenty of them have been granted protected designation of origin status. In the 8th century BCE, Homer described a cheese thought to be the ancestor of feta, the main cheese manufactured in Greece from the ancient times until today. Meanwhile, various cheese types evolved through the centuries, and almost every area in Greece has its own cheesemaking tradition. Some cheese varieties are local, handcrafted products whose production has been handed down from generation to generation, and without interest in their continued production, these varieties will disappear. Other local varieties are made at small factories from pasteurized milk and commercial rennet and starter and are very different from the traditional versions. However, some milk producers still make their cheeses at home or at small dairies from raw milk, without any starter, or sometimes from thermized milk, with traditional yogurt as the starter. Their cheeses are the basis for the information presented in this review.

Selection of dominant NSLAB from a mature traditional cheese according to their technological properties and in vitro intestinal challenges.

Isolates (47) of lactobacilli from 5 different productions of Melichloro cheese were examined for potential use as adjunct cultures. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins classified 29 isolates as L. paraplantarum and 18 as L. paracasei subsp. paracasei. Randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis differentiated the L. paraplantarum and L. paracasei subsp. paracasei isolates at strain level and both, RAPD analysis and whole-cell protein profiling provided useful information about the diversity of nonstarter lactic acid bacteria (NSLAB) in the different cheese productions. The isolates were slow acidifiers and about 70% of them degraded, preferentially α(s)-casein. The amounts of amino acids accumulated in the milk increased with the incubation time. A similar enzyme profile was exhibited by strains of both species, except for α-mannosidase and α-fucosidase, which were not detected in the L. paracasei subsp. paracasei strains. All strains grew in the presence of bile at 0.3% and the majority was able to withstand pH 2.5 and pancreatin at 0.1%. Moreover, all strains reduced cholesterol in vitro, with higher removal ability recorded for strains of L. paraplantarum. A narrow spectrum of antibacterial activity was recorded for 88% of the strains. Selected isolates with appropriate technological and interesting in vitro intestinal challenges could be used as adjuncts and deserve further studies.

Differentiation of lactococci from 2 Greek cheeses with protected designation of origin by phenotypic criteria and RAPD-PCR.

Seventy-six lactococci isolates from 2 protected designation of origin (PDO) cheeses were studied for their acidification ability, proteolytic activity, and inhibitory activities as well as their intraspecies characterization by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Fifty-two of them were characterized as Lactococcus lactis subsp. lactis by the SDS-PAGE of whole-cell proteins. The test strains increased the amount of acid in milk from 6 to 24 h as well as the quantities of amino acids on incubation for 4 d. The majority of the isolates degraded preferentially αs-casein. The isolates from Feta differed from those of Graviera Kritis in respect of ß-casein degradation. This fragment was either not degraded or underwent a small degradation by lactococci from Feta. A stronger intensity of acidification for the isolates from Feta and a higher casein breakdown ability for those from Graviera Kritis were also recorded. Lactococci from Feta and Graviera Kritis inhibited, preferentially, the growth of Escherichia coli O157:H7, and Yersinia enterocolitica, respectively. A high heterogeneity among the isolates according to RAPD-PCR was determined, as well as grouping of the isolates according to their source of isolation. Selected isolates from each cheese could be used as starters to make either Feta or Graviera Kritis.

Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains.

Twelve lactobacilli previously isolated from newborn infants' gastrointestinal tract and Feta cheese were further characterized by pulse field gel eletrophoresis (PFGE). All strains exhibited distinct PFGE genotypic patterns with the exception of DC421 and DC423 strains possessing identical patterns. The strains DC421, 2035 and 2012 were found to posses certain cell surface traits such as hydrophobicity, autoaggregation and/or high adhesive capacity suggesting potential immunomodulatory activity. However, application of the dorsal mouse air pouch system revealed that only the DC421, DC429 and 2035 strains exhibited strong immunostimulatory activity such as increased chemotaxis of polymorphonuclear (PMN) cells in association with increased phagocytosis and cytokine production. The same strains also induced immunomodulatory activity in the gut associated lymphoid tissue in mice in the absence of any inflammatory response. All strains induced IgA production while reduced TNFalpha production by small intestine cells. The strains DC421 and DC429 exerted their effect on the intestine through Toll-like receptor TLR2/TLR4/TLR9 mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-gamma), interleukin (IL)-5, IL-6 and IL-10 are included. The strain 2035 induced similar cytokine profile through the synergy of TLR2/TLR4. This study further supports the eligibility of the air pouch model to discriminate presumptive probiotic Lactobacillus strains exhibiting immunostimulatory activity in the gut. Furthermore, evidence is provided that the cell surface traits examined may not be the only criteria but an alternative and important component of a complex mechanism that enables a microorganism to interact with the host gut to exert its immunoregulatory activity.

Molecular characterization of low-level vancomycin-resistant enterococci found in coastal water of Thermaikos Gulf, Northern Greece.

Enterococcus includes species that may pose emerging health risks and has been used as biomarkers for environmental contamination while little is known concerning their occurrence in marine water. Classification of enterococci in environmental samples can be problematic and requires polyphasic taxonomy. In this study, we investigated the presence of vancomycin-resistant enterococci (VRE) in the inner bay of Thermaikos Gulf in Northern Greece. Based on physiological and biochemical criteria, 121 presumptive enterococcal strains were identified. High-level VRE were undetectable in seawater and only 35 vancomycin gene-negative strains possessed low-level vancomycin resistance. Genotyping by pulsed field gel electrophoresis (PFGE) proved to be more reliable for marine enterococcal discrimination and revealed distinguished characteristics of the seawater enterococci, indicating high genetic diversity. Random amplified polymorphic DNA-PCR (RAPD-PCR) was unable to separate distinct species analyzed in this study. This study indicates the need of polyphasic taxonomy for seawater enterococcal species' identification and provides information for future biomonitoring programs of Thermaikos Gulf.

Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristics and vinification results.

Xynomavro and Muscat Hamburg grapes were fermented spontaneously, producing red and white wine, respectively. The indigenous yeast flora, isolated at three phases of these fermentations, was studied. One hundred and ten strains were isolated, eighty-four of which were identified as Saccharomyces cerevisiae. Six of these strains were selected for their satisfactory oenological properties to be tested as starters in wine fermentations. The quality of the produced wines was evaluated after determination of their aromatic compounds and sensory analysis. S. cerevisiae strain BT4, as well as the AM4+BM3 combination, were considered the best as they produced wines characterized by fruity aromas and were highly accepted among panelists.