Unpasteurised commercial boza as a source of microbial diversity

• The microbiota of three Bulgarian boza was investigated.
• Culture-dependent and -independent methods were applied.
• Boza proved to be a source of biodiversity.
• Microbial species previously undetected in this cereal-based drink were found.

Boza is a cereal-based fermented beverage widely consumed in many countries of the Balkans. The aim of this study was to investigate the microbiota of three Bulgarian boza samples through a combination of culture-dependent and -independent methods with the long-term objective of formulating a multi-strain starter culture specifically destined for the manufacture of new cereal-based drinks. The isolation campaign for lactic acid bacteria (LAB) allowed the identification of Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus coryniformis, Lactobacillus buchneri, Pediococcus parvulus and members of the Lactobacillus casei group. Concerning yeasts, the following isolates were identified: Pichia fermentans, Pichia norvegensis, Pichia guilliermondii (synonym Meyerozyma guilliermondii) and Torulaspora spp. A high intra-species diversity was revealed by Randomly Amplified Polymorphic DNA (RAPD) analysis. In parallel, microbial DNA was directly extracted from the three boza samples, and portions of the rrn operons were analysed through Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The molecular fingerprinting partially confirmed the results of culturing. Among LAB, the species Weissella confusa, Weissella oryzae, Leuconostoc citreum, Lactococcus lactis, Pediococcus parvulus and Pediococcus ethanolidurans were detected together with members of the Lb. casei group. Among the yeasts, the species P. fermentans, M. guilliermondii, Galactomyces geotrichum and Geotrichum fragrans were found. The overall results confirmed boza as having a rich and heterogeneous biodiversity both in terms of species and genetically diverse strains, thus encouraging its exploitation for the isolation and future technological characterisation of cultures to be selected for the manufacture of innovative cereal-based drinks.