Diversity and dynamics of bacterial and fungal communities in cider for distillation

Abstract

Bacterial and fungal population dynamics in cider for distillation have so far been explored by culture-dependant methods. Cider for distillation can be produced by the spontaneous fermentation of apples that do not undergo any intervention during the process. In this study, cider microbiomes extracted from six tanks containing ciders for distillation from four producers in Normandy were characterized at three main stages of the fermentation process: fermentation Initiation (I), end of the alcoholic Fermentation (F) and end of the Maturation period (M). Cider samples were subjected to Illumina MiSeq sequencing (rRNA 16S V1-V3 and ITS1 region targeting) to determine bacterial and fungal communities. Yeasts (YGC), Zymomonas (mZPP) and lactic acid bacteria selective media (mMRS, mMLO, mPSM) were also used to collect 807 isolates. Alcoholic levels, glycerol, sugar content (glucose, fructose and sucrose), pH, total and volatile acidity, nitrogen, malic and lactic acid contents were determined at all sampling points. Alpha diversity indexes show significant differences (p < 0.05) in microbial populations between I, F and M. Fungal communities were characterized by microorganisms from the environment and phytopathogens at I followed by the association of yearsts with alcoholic fermentation like Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora, Candida). A maturation period for cider leads to an increase of the Dekkera/Brettanomyces population, which is responsible for off-flavors in cider for all producers. Among bacterial communities, the genera community associated to malolactic fermentation (Lactobacillus sp., Leuconostoc sp., Oenococcus sp.) was the most abundant at F and M. Acetic acid bacteria such as Acetobacter sp., Komagataeibacter sp. and Gluconobacter sp. were also detected during the process. Significant differences (p < 0.05) were found in fungal and bacterial populations between the four producers and during the fermentation process. The development of microorganisms associated with cider spoilage such as Zymomonas mobilis, Lactobacillus collinoides or Brettanomyces/Dekkera sp. was anticipated by a metagenomic approach. The monitoring of microbial diversity via high throughput sequencing combined with physical-chemical analysis is an interesting approach to improve the fermentation performance of cider for distillation and therefore, the quality of Calvados.

Keywords: 16S and ITS metagenomics; Apple fermentation; Cider for distillation; Dekkera/Brettanomyces; Lactic acid bacteria; Microbial diversity.