N. Tzanetakis

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

C. ANDRIGHETTO, E. PSOMAS, N. TZANETAKIS, G. SUZZI and A. LOMBARDI.2000In the present work randomly amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD–PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty‐two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.

Microbiological characteristics of Batzos, a traditional Greek cheese from raw goat's milk

The changes of microbial flora in Batzos, raw goats milk cheese, were studied during ripening, throughout the whole lactation season in nine cheese batches manufactured three in each, winter, spring and summer. High counts of Enterobacteriaceae and coliforms were recorded early in ripening, but their levels decreased significantly (P<0.05) on ripening and storage. Lactic acid bacteria predominated over the other microbial groups throughout ripening, during the whole lactation season. It seems possible, that high NaCl content of the cheese was the main agent that regulated the microbial survival. No significant differences were observed in the counts related to the season when the cheeses were ripened and stored. However, the season affected the composition of the lactic microflora. Thus, in winter enterococci were abundant, while in spring and summer lactobacilli isolates were found more frequently. Lactococcus lactis subsp lactis was the most frequently isolated species in the cheese and its use as starter seems promising.

Microbiological characteristics of Anevato: a traditional Greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram‐negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log10 cfu g−1 than counts in curd made in January, and were lower by about 2·5 log10 cfu g−1 than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat‐treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.

Some probiotic properties of yeast isolates from infant faeces and Feta cheese

Yeast isolates from infant faeces and Feta cheese were characterized to species level by phenotypic criteria, Randomly Amplified Polymorphic DNA (RAPD)-PCR and mitochondrial DNA (mt-DNA) restriction analysis. Results suggested that there is a good agreement between phenotypic characterization of yeasts and RAPD-PCR at species level; in addition, RAPD-PCR as well as mt-DNA restriction analysis provided good discrimination at strain level. Some technological and probiotic properties of selected strains were also investigated. The test strains exhibited lipolytic and proteolytic activities. They also tolerated low pH and survived satisfactory in gastric juice in vitro as well as in the presence of bile. In general, the isolates from faeces were more resistant to low pH and bile than those from Feta cheese. Selected strains could be used as starter supplements for industrial fermentations.

Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening.

Thirty‐two Lactobacillus plantarum strains isolated from Feta cheese throughout ripening were studied for their phenotypic characteristics, protein profile of cell‐free extracts, enzyme profiles, plasmid profiles, proteolytic and acidifying abilities and ability to grow at low pH and in the presence of bile. Results showed that some biotechnologically important characteristics, such as acidifying and proteolytic activities, can differ between strains. In addition, different plasmid profiles suggest the presence of different Lact. plantarum strains in Feta cheese throughout ripening. The results suggest the possibility of choosing strains with specific biotechnologically interesting properties.

The quality of white-brined cheese from goat's milk made with different starters

Microbiological assessment, biochemical studies and organoleptical evaluation of cheese made from pasteurized goats milk with four different starter cultures were compared. Cheese made with starter B ( Lactococcus lactis subsp. lactis + Lactobacillus casei subsp. casei + Leuconostoc mesenteroides subsp. cremoris 6:2:2), C ( Lact. lactis subsp. lactis + Lb. casei subsp. casei + Enterococcus durans 6:2:2) or D ( Lact. lactis subsp. lactis + Lb. casei subsp. casei + E. durans + Leuc. mesenteroides subsp. cremoris 6:2:1:1) had lower ( P Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus 1:1) and this coincided with higher ( P P P E. durans in the fresh cheese. Cheeses made with starter C or D were graded higher than cheese made with starter A or B. These results suggest that mesophilic starter cultures concurrently used with E. durans might be considered appropriate for the manufacture of white-brined cheese from pasteurized goats milk.

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS‐PAGE of whole cell proteins and rRNA‐targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS‐PAGE and rRNA‐targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.

Probiotic and Technological Properties of Enterococci Isolates from Infants and Cheese

Abstract In the present study 16 enterococcal strains isolated from infant feces and/or Feta cheese were tested for their ability to metabolize in milk and resist specific conditions of the gastrointestinal tract, in order to finally select interesting strains for further studies, for their evaluation as dietary adjuncts. The strains were characterized by phenotypic criteria (API ID 32 STREP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whole-cell protein analysis by SDS-PAGE confirmed the taxonomic allocation of 14 strains suggested by phenotypic criteria as E. durans (4 strains), E. faecalis (3 strains), and E. faecium (7 strains). Two strains biochemically characterized as E. faecalis were assigned to other species by SDS-PAGE. All strains grew well in bile concentrations 0–1%. The strains were also able to withstand low pH (3.0) values for 4 h, eventhough for some strains a reduction by ∼1–2 log10 cfu/mL was observed. Most of the strains produced tyramine and all of them were susceptible to vancomycin. In addition, six out of seven E. faecium strains from either feces or cheese were β-haemolytic. The strains exhibited antagonistic activities towards enterococci, lactic acid bacteria, and Cl. sporogenes. A considerable phenotypic diversity was found among the test strains concerning their acidifying and proteolytic activities. Results of this study give useful information for the selection of appropriate strains with interesting probiotic and technological properties.

Probiotic and Technological Properties of Facultatively Heterofermentative Lactobacilli from Greek Traditional Cheeses

Nineteen isolates of facultatively heterofermentative lactobacilli from Feta, Graviera, and Kasseri cheeses were identified by SDS-PAGE of whole-cell proteins as L. paracasei subsp. paracasei (12 strains) and L. plantarum (7 strains) and differentiated at strain level by RAPD-PCR. Properties of technological interest, such as acidification ability, proteolytic activity, and enzyme activities, were also studied. The test strains exhibited a low acidification activity, with significant interstrain differences after growth in milk for 24 h. They were also characterized by different casein breakdown ability, with around 50% of them accumulating amino acids at low amounts in the milk. Lactobacilli isolates differed in respect of enzyme activities, with β-galactosidase being the strongest activity found. Their probiotic potential was evaluated with in vitro studies on the resistance to low pH, bile salts, and pancreatin. The isolates from Feta showed a better survival than those from Kasseri and Graviera at low pH and viable cells were detected even after 3 h at pH 2.0. All strains tolerated bile salts at 0.3% and retained viability in the presence of pancreatin at 0.1%. Different patterns of antibacterial activities were recorded. The strains inhibited preferentially LAB species and some of them clostridia, E. coli O44 and B. cereus. Distinguished strains are promising probiotic candidates as adjuncts and deserve further studies.

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 β-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 β-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.