Improvement of traditional vinegar production by selected acetic acid bacterium strain: Acetobacter pasteurianus as acetification starter

Recent microbiological studies investigated the acetic acid bacteria microflora of traditional balsamic vinegar (TBV) highlighting the occurrence of Gluconacetobacter europaeus as widespread indigenous species, followed by Acetobacter pasteurianus, Acetobacter aceti and Acetobacter malorum1,2. However no correlation between occurring species and quality of product have been examined. In this study a selected starter culture (SSC) was designed, implemented at laboratory scale and applied to vinegar factory by scale up procedure. A. pasteurianus strain AB0220 was selected as SSC on the basis of phenotypical and technological traits suitable for TBV must oxidation. The SSC was implemented through three stages: starting steps in laboratory conditions (Stage 1); scale-up performed through a tanks system (Stage 2) and a barrels system (Stage 3) in factory conditions. Main analytical parameters were monitored by pH, titrable acidity, ethanol and soluble solids trend. Moreover molecular identification based on 16S rDNA region analysis (PCR-DGGE and sequencing) and ERIC-PCR were performed respectively to assess species occurrence and evaluate strain persistence during the whole process. In particular the dominant AAB species population was estimated by PCR-DGGE analysis allowing to distinguish two different species groups along the 3 stages. 16S rDNA sequencing confirmed DGGE results, showing high percentage of sequence homology (99 and 100%) with A. pasteurianus at stage 1 and 2 and Ga. europaeus at Stage 3. Finally, ERIC-PCR fingerprinting assay performed at the end of each acetification stage showed an electrophoretic profile similar to that of AB0220 at stage 1 and 2, whereas a different pattern at stage 3. This result supports DGGE data suggesting a change in the population during the 3 acetification stages. On the basis of these evidences, our hypothesis is that the persistence of inoculated strain during stages 1 and 2 was assured by the scale-up procedure. New must was periodically added to increase the SSC volume and microbial population was not exposed to constant effect of acetic acid. Instead, the static conditions of stage 3 resulted in a constant increase of acetic acid concentration, negatively affected AB0220 growth. In this environment, indigenous Ga. europaeus cells, which is less sensitive to physiological stress caused by acetic acid3, found optimal growth conditions to become dominant. In this study for the first a SSC for TBV production was implemented and applied at factory scale. Results demonstrated that selected A. pasteurianus strains are suitable to start the acetification of TBV ensuring the acetification course in unsuitable conditions for other AAB such as Ga. europaeus. On the contrary, Ga. europaeus strains are able to oxidise cooked must in presence of constant of acetic acid concentration corresponding to the final stages of TBV oxidation. Results suggest that the introduction of SSC could be a valid innovation in TBV production, contributing to safety and quality of the product and improving the reliability and stability of technological process.