Pyrosequencing vs culture dependent identification to analyze LAB population associated to Chicha, a traditional maize-based fermented beverage from Northwestern Argentina

AZNAR, ROSA; ELIZAQUÍVEL, PATRICIA; YEPES, ALBA; JIMENEZ, MARÍA EUGENIA; ARISTIMUÑO, CECILIA; PEREZ, ALBA; COCCONCELLI, PS; VIGNOLO, G

Valencia

Congreso; ECCO XXXIII Molecular Taxonomy: from biodiversity to biotechnology; 2014

Traditional fermented foods and beverages are produced by natural fermentation involving mixed populations of indigenous microorganisms. Among them lactic acid bacteria (LAB) are relevant players in the process. Chicha is a traditional maize-based fermented alcoholic beverage produced following artisanal and ancestral procedures in the Andean regions. In this study the diversity of LAB associated with chicha fermentation was analysed using culture-dependent and culture-independent procedures to assess genus/species composition. To that aim, samples from two chicha productions were obtained from local producers at Maimará (chicha M) and Tumbaya (chicha T) in northwestern Argentina, each exhibiting particular manufacturing procedures. Samples corresponding to 10 production steps were analyzed by pyrosequencing of the V3-V5 region in the 16S rRNA gene to obtain a snapshot of the global bacterial population. Sequence analysis and taxonomic assignment was performed using the Ribosomal Database Project II (RPD Naïve Bayesian rRNA Classifier Version 2.4) at 97% sequence similarity with a confidence of 90%. Among LAB population, six genera were identified in both chichi production: Enterococcus, Lactococcus, Streptococcus, Weissella, Leuconostoc and Lactobacillus while Pediococcus was only detected in chicha M. At species level, the highest diversity was observed for Lactobacillus from chicha M and T, and Enterococcus from chicha T. Predominant LAB species in chicha M were Lb. plantarum, Lb. rossiae, Lc. lactis and W. viridescens whereas in chicha T were E. ratti and Lc. mesenteroides. In parallel, presuntive LAB isolated (chicha M: 146 and chicha T: 246) recovered of the same samples were identified by ISR-PCR and RAPD-PCR profiles, species specifics PCR and 16S rRNA gene sequencing. Results showed most of the isolates from chicha M as belonging to Leuconostoc genus (Lc. mesenteroides and Lc. lactis) although Lactobacillus, Weissella and Enterococcus were also present. In contrast, chicha T exhibited the presence of Enterococcus and Leuconostoc, E. faecium being the most representative species during fermentation. Members of genera Lactococcus, Pediococcus and Streptococcus were only detected by pyrosequencing in chicha M as with Weissella, Lactococcus, Lactobacillus and Streptococcus in chicha T. Pyrosequencing revealed the highest species diversity for Lactobacillus and Enterococcus in chicha M and T samples, respectively. Of them, around 5% of the Lactobacillus species were found by culturing in chicha M, but none in chicha T. In conclusion, culture-dependent approach showed marked differences in LAB diversity from both chicha productions, whereas pyrosequencing, although provided a more complete inventory of bacteria diversity, revealed similar profiles of genus/species in both productions, thus pointing to the species which may drive the fermentation process.