Thermization widely used to improve the microbiological quality of raw milk intended for traditional cheese making also inactivates a major part of the beneficial mesophilic LAB biota, particularly wild Leuconostoc spp. In this study, thirteen Leuconostoc mesenteroides isolates from counterpart raw/thermized (65 °C; 30 sec) milks of native Epirus sheep breeds, previously identified by 16S rRNA gene sequencing and at the subspecies according to their multiplex-PCR (rpoB, araA, dsr, sorA) profiles, were biochemically characterized. The isolates formed seven biotypes, with only one assigned to the subspecies mesenteroides and none to cremoris. Five biotypes shared an atypical dextranicum (dsr/sorA) genotype and one biotype seemed novel as it was genomically assignable to the subspecies jonggajibkimchii (araA/dsr). Six biotypes comprised strains with a strong-positive or positive leucine arylamidase activity, but their valine and cystine arylamidase activities were weak or absent. All except two isolates were β-galactosidase-positive whereas only two typical mesenteroides isolates from thermized milk were α-galactosidase-positive. All isolates fermented lactose, but were weak acidifiers in skimmed milk; 46% were dextran-negative. None was β-hemolytic, or possessed β-glucoronidase, trypsin or α-chymotrypsin activity, or formed histamine or tyramine. Excluding their inherent resistance to vancomycin, all biotypes comprised antibiotic susceptible strains. Only one strain was resistant to ampicillin, and five strains were partially susceptible to ciprofloxacin. Two raw sheep milk strains, KFM7 (atypical dextranicum; relatively more aciduric and proteolytic in vitro) and KFM9 (jonggajibkimchii; bacteriocinogenic in milk) previously selected and applied as adjuncts to produce a novel semi-industrial Kefalotyri cheese type from thermized Epirus sheep milk were confirmed to be the most promising biotypes for onsite commercial use in cheese production.
Citation: John Samelis, Athanasia Kakouri. Biotype-based validation of wild Leuconostoc mesenteroides strain genotypes from raw/thermized milks of native Epirus sheep breeds selected and preliminarily applied as cheese adjuncts[J]. AIMS Microbiology, 2026, 12(4): 865-901. doi: 10.3934/microbiol.2026037
Thermization widely used to improve the microbiological quality of raw milk intended for traditional cheese making also inactivates a major part of the beneficial mesophilic LAB biota, particularly wild Leuconostoc spp. In this study, thirteen Leuconostoc mesenteroides isolates from counterpart raw/thermized (65 °C; 30 sec) milks of native Epirus sheep breeds, previously identified by 16S rRNA gene sequencing and at the subspecies according to their multiplex-PCR (rpoB, araA, dsr, sorA) profiles, were biochemically characterized. The isolates formed seven biotypes, with only one assigned to the subspecies mesenteroides and none to cremoris. Five biotypes shared an atypical dextranicum (dsr/sorA) genotype and one biotype seemed novel as it was genomically assignable to the subspecies jonggajibkimchii (araA/dsr). Six biotypes comprised strains with a strong-positive or positive leucine arylamidase activity, but their valine and cystine arylamidase activities were weak or absent. All except two isolates were β-galactosidase-positive whereas only two typical mesenteroides isolates from thermized milk were α-galactosidase-positive. All isolates fermented lactose, but were weak acidifiers in skimmed milk; 46% were dextran-negative. None was β-hemolytic, or possessed β-glucoronidase, trypsin or α-chymotrypsin activity, or formed histamine or tyramine. Excluding their inherent resistance to vancomycin, all biotypes comprised antibiotic susceptible strains. Only one strain was resistant to ampicillin, and five strains were partially susceptible to ciprofloxacin. Two raw sheep milk strains, KFM7 (atypical dextranicum; relatively more aciduric and proteolytic in vitro) and KFM9 (jonggajibkimchii; bacteriocinogenic in milk) previously selected and applied as adjuncts to produce a novel semi-industrial Kefalotyri cheese type from thermized Epirus sheep milk were confirmed to be the most promising biotypes for onsite commercial use in cheese production.
This research received no external funding. All the consumables used in this study were leftover stock materials from the research projects BIOTRUST (T1EDK-00968) and ProMedFoods (ARIMNet 2, ID 9028; Grant No 618127) previously conducted in our laboratory. Dr John Samelis, the present corresponding and submitting author, was the scientific responsible researcher coordinating both previous projects above.
The authors declare no conflict of interest.
Dr John Samelis—Conceptualization, Investigation, Corresponding—Supervision, Compilation, Preparation of first draft, Reviewing, Editing; Athanasia Kakouri—Investigation, Reviewing, Editing.
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Figures(2) / Tables(6)
John Samelis, Athanasia Kakouri. Biotype-based validation of wild Leuconostoc mesenteroides strain genotypes from raw/thermized milks of native Epirus sheep breeds selected and preliminarily applied as cheese adjuncts[J]. AIMS Microbiology, 2026, 12(4): 865-901. doi: 10.3934/microbiol.2026037
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