Magdalini Hatzikamari

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.

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.

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.

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 β-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.

Preservation of pears in water in the presence of Sinapis arvensis seeds: A Greek tradition

In this research, the microbiological and physicochemical changes during preservation of pears in water in the presence of Sinapis arvensis seeds (PWS FL) according to the traditional Greek home food manufacture were studied. Pears preserved in water served as control (PW FL). The growth of lactic acid bacteria (LAB) coming from the pear surface was enhanced in the presence of Sinapis seeds, while Enterobacteriaceae and Gram-negative bacteria declined coincidently with the lower (P<0.05) pH of the PWS FL. LAB predominated over the other microbial groups in the fermentation liquids (FLs) of both systems. All the 49 LAB isolates from one fermentation experiment were identified as Leuconostoc mesenteroides subsp. cremoris by the SDS-PAGE of whole-cell proteins, while RAPD-PCR fingerprinting and partial 16S rRNA sequence determination of selected isolates did not discriminate them at the subspecies level. Fruit preserved in PWS FL had higher titratable or volatile acidity, phenolic compounds or antioxidant capacity as well as lower pH and firmness than the control fruit. All physicochemical parameters of the FLs increased except of the pH which decreased. Coincidently with higher population of LAB in PWS FL the levels of citric, lactic and acetic acid were higher than in control. Oxalic acid and related unknown substances were found at higher levels in PWS FL than the control and may be the agent(s) enhancing the growth of LAB and/or contributing partially to the decline of Enterobacteriaceae. The organoleptic test showed that fruit preserved in PWS FL had better overall acceptance than the control, and that it retained most of the positive traits.