No-culture strategy for tracking AAB and yeasts in low-acidic and high polyphenolic niches

Community of acetic acid bacteria (AAB) and yeasts inhabitslow acidic and high polyphenolic niches such as fermentedbeverages. In this work, we selected kombucha asmodel to optimize a no-culture strategy for tracking thedynamics of AAB and yeasts. The strategy consisted of: i)Genomic DNA extraction optimization, ii) PCR/DGGEsetup and iii) Validation of the assay. For gDNA extractionoptimization, we evaluated the suitability of the followinggDNA preparations for a standard PCR-DGGE assay: 1)CTAB/AAB; 2) CTAB/vinegar; 3) Freeze/bead/CTAB1; 4)Freeze/bead/CTAB2 methods. Different extraction methodsaffected not only the quantity and purity of DNA but alsothe complexity of PCR/DGGE profiling. Method 4 allowedus to obtain suitable DNA for PCR assays, as well as thehighest level of complexity in PCR/DGGE band patternsboth for AAB and yeasts. Moreover changes from exopolysaccharidicto liquid phases did not affect the suitability ofthis method. PCR/DGGE setup was performed consideringthe following variables: DNA template concentration, PCRconditions and specificity, effect of GC-clamp and nestedPCR. For AAB, primers pair WBAC1/WBAC2 enabled tostudy AAB dynamics as well other bacterial groups, whereas341f/518r permitted the detection of members of theGluconacetobacter genus. For yeasts, primers pair U1/U2was more efficient then NL1/LS2 in community profiling ofthe majority of kombucha samples. GC-clamp added toforward primers negatively affected gDNA suitability and anested re-PCR for yeasts and AAB was required. Finally,validation of the assay was carried out during kombuchafermentation following AAB and yeasts dynamics from theinoculum to 12 days of incubation. Within AAB species,Gluconacetobacter xylinus responsible for the exopolysaccharidicnetwork matrix, was mainly detected, whereasZygosaccharomyces spp, Dekkera spp, Pichia spp andSchizosaccharomyces pombe in the case of yeasts.