Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the Leuconostoc genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50 Leuconostoc pseudomesenteroides [Ln. pseudomesenteroides] phages, 4 Ln. mesenteroides phages) and from 3 external phage collections (17 Ln. pseudomesenteroides phages, 12 Ln. mesenteroides phages). All phages belonged to the Siphoviridae family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all Ln. mesenteroides phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all Ln. pseudomesenteroides phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All Ln. mesenteroides and all Ln. pseudomesenteroides phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all Leuconostoc phage types.     

Characterization of Leucocin B-KM432Bz from Leuconostoc pseudomesenteroides Isolated from Boza,and Comparison of its Efficiency to Pediocin PA-1

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII^t _Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.     

Plasmid-Encoded Diacetyl (Acetoin) Reductase in Leuconostoc pseudomesenteroides

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides.     

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> 607 Isolated from Persimmon

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C₁₈ cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH₂-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.     

Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Spontaneous Formation of a Mannitol-Producing Variant of Leuconostoc pseudomesenteroides Grown in the Presence of Fructose

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.     

Identification of the Receptor-Binding Protein in Lytic Leuconostoc pseudomesenteroides Bacteriophages

Two phages, P793 and ΦLN04, sharing 80.1% nucleotide sequence identity but having different strains of Leuconostoc pseudomesenteroides as hosts, were selected for identification of the host determinant gene. Construction of chimeric phages leading to the expected switch in host range identified the host determinant genes as ORF21_P793/ORF23_ΦLN04. The genes are located in the tail structural module and have low sequence similarity at the distal end.     

Characterization and antioxidant activity of an exopolysaccharide produced by Leuconostoc pseudomesenteroides JF17 from juçara fruits (Euterpe edulis Martius)

Juçara palm tree is considered an important species in the Atlantic forest ecosystem and the exploitation of its fruits is a main sustainable strategy adopted to its maintenance. Among several microorganisms in the juçara fruits, were found lactic acid bacteria (LAB) highly capable of synthesizing exopolysaccharides (EPS). Thus, the EPS synthesized by Leuconostoc pseudomesenteroides JF17 was characterized according to its chemical structure, physicochemical properties and antioxidant activity. HPLC analysis showed that the EPS consisted of a homopolysaccharide composed of glucose units, FT-IR spectroscopy revealed a dextran, confirmed by ¹H and ¹³C NMR spectra which showed the composition of the polysaccharide, d-glucose units bound by α-(1→6). The EPS showed high water retention capacity, 507.812 ± 47.471 %, and may be related to the porous structure revealed by SEM images. Thermal analysis indicated that the EPS had excellent thermal stability with degradation temperature of 320.8 °C and melting point at 279.81 °C, with the enthalpy of 106.557 J/g. The EPS also presented sequestration ability on different free radicals tested, indicating high antioxidant activity. The results suggest that L. pseudomesenteroides JF17 might be widely used in the production of dextran which has potential qualities for applications in the food industry.     

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15

Aim: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. Methods and Results: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A‐QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A‐QU 15 were identical to that of leucocin A‐UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C‐terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A‐QU 15, leucocin Q and leucocin N were obtained. Conclusions: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C‐terminal region of leucocin A‐QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A‐QU 15 was not. Significance and Impact of Study: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.     

How water-soluble saccharides drive the metabolism of lactic acid bacteria during fermentation of brewers' spent grain

We proposed a novel phenomic approach to track the effect of short-term exposures of Lactiplantibacillus plantarum and Leuconostoc pseudomesenteroides to environmental pressure induced by brewers' spent grain (BSG)-derived saccharides. Water-soluble BSG-based medium (WS-BSG) was chosen as model system. The environmental pressure exerted by WS-BSG shifted the phenotypes of bacteria in species- and strains-dependent way. The metabolic drift was growth phase-dependent and likely underlay the diauxic profile of organic acids production by bacteria in response to the low availability of energy sources. Among pentosans, metabolism of arabinose was preferred by L. plantarum and xylose by Leuc. pseudomesenteroides as confirmed by the overexpression of related genes. Bayesian variance analysis showed that phenotype switching towards galactose metabolism suffered the greatest fluctuation in L. plantarum. All lactic acid bacteria strains utilized more intensively sucrose and its plant-derived isomers. Sucrose-6-phosphate activity in Leuc. pseudomesenteroides likely mediated the increased consumption of raffinose. The increased levels of some phenolic compounds suggested the involvement of 6-phospho-β-glucosidases in β-glucosides degradation. Expression of genes encoding β-glucoside/cellobiose-specific EII complexes and phenotyping highlighted an increased metabolism for cellobiose. Our reconstructed metabolic network will improve the understanding of how lactic acid bacteria may transform BSG into suitable food ingredients.     

Isolation and Characterization of Bacteriophages from Fermenting Sauerkraut

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.     

High-yield production of mannitol by <Emphasis Type="Italic">Leuconostoc pseudomesenteroides</Emphasis> CTCC G123 from chicory-derived inulin hydrolysate

Chicory is an agricultural plant with considerable potential as a carbohydrate substrate for low-cost production of biochemicals. In this work, the production of mannitol by Leuconostoc pseudomesenteroides CTCC G123 from chicory-derived inulin hydrolysate was investigated. The bioconversion process initially suffered from the leakage of fructose to the phosphoketolase pathway, resulting in a low mannitol yield. When inulin hydrolysate was supplemented with glucose as a substrate for mannitol production in combination with aeration induction and nicotinic acid induced redox modulation strategies, the mannitol yield greatly improved. Under these conditions, significant improvement in the glucose consumption rate, intracellular NADH levels and mannitol dehydrogenase specific activity were observed, with mannitol production increasing from 64.6 to 88.1 g/L and overall yield increase from 0.69 to 0.94 g/g. This work demonstrated an efficient method for the production of mannitol from inulin hydrolysate with a high overall yield.     

Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization

A gene encoding mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) was cloned from Lactobacillus reuteri and expressed in Escherichia coli. The 1,008-bp gene encodes a protein consisting of 336 amino acids, with a predicted molecular mass of 35,920 Da. The deduced amino acid sequence of L. reuteri MDH (LRMDH) is 77% and 76% similar to the MDHs from Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, respectively. The purified recombinant enzyme appears as a single band of 40 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but gel filtration indicates that the native enzyme is a dimer. The optimum temperature for the recombinant enzyme is 37°C, the pH optima for D-fructose reduction and D-mannitol oxidation are 5.4 and 6.2, respectively. The K_m values for NAD (9 mM) and NADH (0.24 mM) are significantly higher than those for NADP (0.35 mM) and NADPH (0.04 mM). The K_m values of LRMDH for D-fructose and D-mannitol are 34 mM and 54 mM, respectively. Contrary to what the enzyme sequence suggests, recombinant LRMDH contains a single catalytic zinc per subunit.     

Antibiotic Susceptibility Profiles of Dairy Leuconostoc,Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.     

Acetaldehyde Utilization by Leuconostoc Species

Single-strain cultures of Leuconostoc dextranicum and L. mesenteroides were found to be capable of utilizing acetaldehyde in both acidified and nonacidified milk cultures at 21 and 30 C. L. dextranicum utilized acetaldehyde more rapidly than L. mesenteroides when the pH of the medium was near that of normal ripened lactic dairy cultures. Acetaldehyde production and utilization varied widely among different two-strain cultures of lactic streptococci and leuconostocs. The concentration of acetaldehyde in ripened single-strain lactic streptococcal cultures was lowered by adding a large inoculum of ripened L. citrovorum culture and continuing incubation at 21 or 5 C.     

Characterization,Identification and Application of Lactic Acid Bacteria Isolated from Forage Paddy Rice Silage

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage.     

DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.     

Bacterial Community Structure and Location in Stilton Cheese

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.     

Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.