Kasseri Cheese Research Articles

The microbiology of Kasseri cheese during the maturation

The microbiology of Kasseri cheese, a Protected Designation of Origin (PDO) cheese of pasta-filata type was studied in order to identify the dominant species and strains that may contribute to the maturation process. Chemical composition and microbiological quality of Kasseri cheese samples from two different dairies during the maturation was studied at 0, 7, 25, 60 and 90 days of the maturation. Lactic acid bacteria and Enterococcus spp. were found to be the dominant microflora in fresh cheese. P. pentosaceous and P. acidilactici, E. hirae, E. faecium, E. durans and E. gallinarium, together with facultatively heterofermentative lactobacilli were found to be the dominant microflora. Since these strains are dominating the microflora of Kasseri cheese during maturation, the enzymic system need to be further studied in order to select the proper strains for adjunct culture in Kasseri cheese.

The microbiology of Kasseri cheese during the maturation

The microbiology of Kasseri cheese during the maturation

Complete Genome Sequence of the Dairy Isolate Streptococcus macedonicus ACA-DC 198

The species Streptococcus macedonicus is associated with the food environment, especially with fermented dairy products. Here we present the complete 2.1-Mb genome sequence of strain ACA-DC 198, which was isolated from naturally fermented Greek kasseri cheese.

Invitro and invivo safety evaluation of the bacteriocin producer Streptococcusmacedonicus ACA-DC 198

Streptococcus macedonicus ACA-DC 198, a bacteriocin producer isolated from Greek Kasseri cheese, was used in a series of in vitro and in vivo experiments in order to evaluate its pathogenic potential. The strain was examined in vitro for haemolytic activity, antibiotic resistance and presence of pathogenicity genes encountered in Streptococcus pyogenes. Subsequently, the strain was orally administered to mice (8.9 log cfu daily), continuously over a period of 12 weeks, in order to ascertain the effects of its long term consumption on animal health and gastric inflammation. S. macedonicus ACA-DC 198 was found to be non-haemolytic and sensitive to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, streptomycin, tetracycline, and vancomycin, with the only resistance observed against kanamycin. PCR amplification and DNA–DNA hybridization did not reveal the presence of any of the S. pyogenes pathogenicity genes examined, namely emm, scpA, hasA, speB, smez2, speJ, sagAB, hylA, ska, speF, speG, slo, hylP2 and mga. In the mouse study, no detrimental effects were observed in the behaviour, general well being, weight gain and water consumption of the animals receiving S. macedonicus ACA-DC 198. Histologic analysis showed no evidence of inflammation in the stomach of the animals receiving S. macedonicus ACA-DC 198, while faecal microbiological analysis revealed that the strain retained its viability passing through the mouse gastrointestinal tract. Finally, no evidence of translocation to the liver, spleen and mesenteric lymph nodes was observed. In conclusion, none of the examined virulence determinants were detected in S. macedonicus ACA-DC 198 and its long term, high dosage oral administration did not appear to induce any pathogenic effect in mice.

Inhibition of Clostridium tyrobutyricum by Streptococcus macedonicus ACA-DC 198 under conditions mimicking Kasseri cheese production and ripening

Three fermentations in skim milk were used to study the effectiveness of the bacteriocin-producing Streptococcus macedonicus ACA-DC 198 strain to inhibit Clostridium tyrobutyricum LMG 1285T spore outgrowth under conditions prevailing during Kasseri cheese production and ripening. In fermentation A, Clostridium spores were used solely; in fermentation B, S. macedonicus ACA-DC 198 and Clostridium spores were used; in fermentation C, a commercial starter culture and Clostridium spores were used. The temperature program applied was similar to that of Kasseri cheese production and ripening. The presence of macedocin, the bacteriocin produced by S. macedonicus ACA-DC 198, was confirmed in fermentation B. The results showed that macedocin was able to inhibit the outgrowth of Clostridium spores, since significantly higher inhibition in spore outgrowth was detected in fermentation B than in fermentation C.

Characterization of the gene cluster involved in the biosynthesis of macedocin, the lantibiotic produced byStreptococcus macedonicus

Streptococcus macedonicus ACA-DC 198, a food-grade isolate from naturally fermented Greek Kasseri cheese, produces a lantibiotic named macedocin that has been previously purified and characterized. In the present study, a 15,171 bp region in the S. macedonicus ACA-DC 198 chromosome, containing the biosynthetic gene cluster of macedocin, has been sequenced. This region consists of 10 ORFs, which correspond to the genes (mcd genes) involved in macedocin biosynthesis, regulation and immunity. The mcd genes are organized in two operons and their role is predicted on the basis of similarities to genes of known lantibiotics. Compared with its closest match, the streptococcin A-FF22 gene cluster, the macedocin one contains an additional structural gene and an insertion sequence between the regulatory and the biosynthetic operons.

The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production

Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of all microbial groups examined initially increased, but declined after Baski scalding and remained practically stable up to day 90. The presence of S. macedonicus was confirmed until the end of ripening (Cheeses B and C); this was also the case for the bacteriocin produced by S. macedonicus ACA-DC 198. X-propyl-dipeptidyl-aminopeptidase activity was detected in all cheeses after day 15. The physicochemical characteristics of the mature Cheese C were in agreement with those defined by the Greek legislation. The sensory properties of all mature cheeses corresponded with the ones characterizing the traditional Kasseri cheese. It was concluded that S. macedonicus ACA-DC 198 can be used as an adjunct in Kasseri cheese production.

Macedocin, a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198.

Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.

Effect of technological parameters on the characteristics of kasseri cheese made from raw or pasteurized ewes’ milk

Two groups of kasseri cheese (pasta filata type) were manufactured from raw or pasteurized ewes’ milk, without starter cultures. Cheeses of each group were divided into two subgroups: the first was ripened and stored at 4°C and packaged in plastic film; the second ripened and stored at 15°C and coated with paraffin wax. Milk pasteurization and technological parameters had a significant effect on the pH ( P < 0.05), while only technological parameters had an effect on the total solids content. At day 120, the range of mean cfu/g counts for the mesophilic aerobic flora was 9.5 × 107−1.4 × 108; for the thermophilic streptococci, the range was 2.6 × 107−7.6 × 107; and for the thermophilic bacilli, 9.8 × 106−1.7 × 107. Changes in the N fractions became significant after 30 days of ripening. For mature 120‐day‐old cheeses, the percentage of total N soluble at pH 4.6 was 22.7%–22.9% in raw milk cheeses and 19.0%–21.7% in pasteurized milk cheeses. The percentage of total N soluble at 12% TCA was 10.1%–12.2% in raw milk cheeses and 7.3%–11.5% in pasteurized milk cheeses; the percentages of total N soluble at 5% PTA were 3.1%–4.0% and 2.6%–3.6%, respectively. The residual αs‐casein percentages at day 120 ranged between 63% and 78% of the respective area at day 1; the residual β‐casein ranged between 67% and 75%. There were some characteristic differences in the reverse phase‐HPLC peptide profiles of the four cheeses. In general, the effect of the different ripening conditions was more pronounced in cheeses made from pasteurized milk.

Production of processed cheese using kasseri cheese and processed cheese analogues incorporating whey protein concentrate and soybean oil

A control processed cheese (A) made mainly from kasseri cheese (60%) without whey protein concentrate or soybean oil, and three other cheese products B, C and D containing increasing amounts of whey protein concentrate (UF) and soybean oil were manufactured simultaneously. All the cheeses were produced to contain 50–51% moisture and 53–54% fat‐in‐dry‐matter and were submitted to microbiological, physicochemical, theological and organoleptic tests 1 day after production and after 90 days in cold storage under vacuum. The mesophilic and psychrotrophic microflora of all the cheeses was very low; coliforms were not found. All the cheeses differed significantly in their content of total protein, soluble protein, lactose, ash, acidity (ADV) and in the oxidation of unsaturated fatty acids (cheese D). In contrast, no significant differences in pH, moisture or fat were noted because of the standardization of the blends. Rheological tests of the products indicated that there were marked differences in hardness, adhesiveness, elasticity, gumminess and chewiness. The cheeses were subjected to sensory analysis and showed differences in flavour, texture and spreading ability on day 1 and, moreover, in appearance after 90 days.

Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese.

A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production. All strains were found to be moderate acidifiers in milk. Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin. Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide. Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate. Only one showed activity up to palmitate; this was also the most active strain against tributyrin. Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides. Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp. shermanii. Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine.

Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: characterization and specificity for beta-casein.

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.

Combined effects of concentrated thermophilic and mesophilic cultures and conditions of curd acidifications on the manufacture and quality of kasseri cheese

The effect offour process variables on the biological acidification of curd to be usedfor the manufacture of kasseri cheese was examined. The variables were (i) concentrated starter cultures: a thermophilic mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgar‐icus compared with a blend of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei subsp casei, and (ii) acidification temperatures: 30 and 40° C for the thermophilic cultures and 20 and 30°C for the mesophiles. The use of concentrated starter cultures enabled the cheese to be made in one day as compared with three in the traditional system usually using raw milk and no culture. The mesophilic blend combined with a lower scalding temperature (35°C) gave a higher yield than the alternative culture, and the cheeses were higher in total solids, acidity andsalt; the same cheeses were also harder and less elastic. The use of mesophilic cultures and a low acidification temperature (20° C) is recommended.

Identification of streptococci from Greek Kasseri cheese and description of Streptococcus macedonicus sp. nov.

Taxonomic studies were performed on some Streptococcus-like organisms isolated from naturally fermented Greek Kasseri cheese. By SDS-PAGE analysis of whole-cell proteins the group was found to be quite different from Streptococcus thermophilus. Comparative 16S and 23S rRNA sequence analyses showed that the isolates represent a new species within the genus Streptococcus, where they are most closely related to the Streptococcus bovis cluster. On the basis of these phylogenetic results and some phenotypic differences, a new species, Streptococcus macedonicus, is proposed. The type strain is ACA-DC 206.