Carla Jara

Population dynamics of acetic acid bacteria during traditional wine vinegar production.

The population dynamics of acetic acid bacteria in traditional vinegar production was determined in two independent vinegar plants at both the species and strain level. The effect of barrels made of four different woods upon the population dynamics was also determined. Acetic acid bacteria were isolated on solid media and the species were identified by RFLP-PCR of 16S rRNA genes and confirmed by 16S rRNA gene sequencing, while strains were typed by ERIC-PCR and (GTG)(5)-rep-PCR. The most widely isolated species was Acetobacter pasteurianus, which accounted for 100% of all the isolates during most of the acetification. Gluconacetobacter europaeus only appeared at any notable level at the end of the process in oak barrels from one vinegar plant. The various A. pasteurianus strains showed a clear succession as the concentration of acetic acid increased. In both vinegar plants the relative dominance of different strains was modified as the concentrations of acetic acid increased, and strain diversity tended to reduce at the end of the process.

Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods

Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

Effect of wood type and thickness on acetification kinetics in traditional vinegar production

Traditional vinegar production is a lengthy process which implies high operational risks and jeopardizes the organoleptic characteristics of the final product. In an effort to solve these problems without changing the traditional model, we modified the wood type and thickness of vinegar barrels. We acetified in triplicate in barrels made of acacia, cherry, chestnut, and oak and in three wood thicknesses (15, 20, and 25 mm) in two different vinegar plants. The operating volume was set at 60 L. Reducing wood thickness improved neither maximum acetification velocity or the total length of the process, and in some cases even worsened them. The process took longer in oak barrels than in other types of wood barrel in one of the vinegar plants. Therefore, the choice of wood is a parameter to be considered in the wine vinegar production.

Genome Sequences of Three Oenococcus oeni Strains Isolated from Maipo Valley, Chile

ABSTRACT Oenococcus oeni is part of the microbial terroir involved in wine production. Here, we present three genome sequences of O. oeni strains isolated from spontaneous malolactic fermentation of cultivar Cabernet Sauvignon Maipo Valley, Chile.

Microbial Terroir in Chilean Valleys: Diversity of Non-conventional Yeast.

In this study, the presence of non-conventional yeast associated with vineyards located between latitudes 30°S and 36°S was examined, including the valleys of Limarí, Casablanca, Maipo, Colchagua, Maule, and Itata. The microbial fingerprinting in each valley was examined based on the specific quantification of yeast of enological interest. Grape–berries were sampled to evaluate the presence and load of non-conventional yeast with enological potential, such as Metschnikowia, Hanseniaspora, Torulaspora, Debaryomyces, Meyerozyma, and Rhodotorula. These yeasts were present in all vineyards studied but with varying loads depending on the valley sampled. No identical fingerprints were observed; however, similarities and differences could be observed among the microbial profiles of each valley. A co-variation in the loads of Metschnikowia and Hanseniaspora with latitude was observed, showing high loads in the Casablanca and Itata valleys, which was coincident with the higher relative humidity or rainfall of those areas. Non-conventional yeasts were also isolated and identified after sequencing molecular markers. Potentially good aromatic properties were also screened among the isolates, resulting in the selection of mostly Metschnikowia and Hanseniaspora isolates. Finally, our results suggest that microbial terroir might be affected by climatic conditions such as relative humidity and rainfall, especially impacting the load of non-conventional yeast. In this study, the microbial fingerprint for yeast in Chilean vineyards is reported for the first time revealing an opportunity to study the contribution of this assembly of microorganisms to the final product.

Analysis of acetic acid bacteria by different culture-independent techniques in a controlled superficial acetification

Three molecular techniques [denaturing gradient gel electrophoresis (DGGE-PCR), restriction fragment length polymorphism of the 16S rRNA gene amplicon (RFLP-PCR 16S rRNA) and real-time PCR (RT-PCR) with SybrGreen and with specific TaqMan-Minor Groove Binder (MGB) probes] were used to identify and monitor acetic acid bacteria (AAB) species during a controlled acetification. This process was initiated by seeding a starter culture comprising a mixture of one strain each of Acetobacter pasteurianus, Gluconacetobacter europaeus and Gluconacetobacter hansenii. Analysis at the species level indicated that the population of A. pasteurianus increased quickly, subsequently acquiring a dominant position, whereas the other two species gradually disappeared. All three methods confirmed this result. When the total AAB population was estimated, the results obtained based on summing the three species by TaqMan-MGB RT-PCR, total AAB RT-PCR and the direct microscopic count method were similar. Using TaqMan-MGB RT-PCR we were able to detect species with populations 3 log units lower than that of the major species and which could not be detected by other methods.

The rapid identification of lactic acid bacteria present in Chilean winemaking processes using culture-independent analysis

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S ribosomal RNA (rRNA) genes was developed to identify lactic acid bacteria (LAB) that are commonly present in winemaking processes (Oenococcus, Pediococcus, Lactobacillus, and Leuconostoc). This culture-independent approach revealed the presence of Oenococcus in the spontaneous malolactic fermentation in industrial Chilean wines.

Oenococcus oeni in chilean red wines: technological and genomic characterization

The presence and load of species of LAB at the end of the malolactic fermentation (MLF) were investigated in 16 wineries from the different Chilean valleys (Limarí, Casablanca, Maipo, Rapel, and Maule Valleys) during 2012 and 2013, using PCR-RFLP and qPCR. Oenococcus oeni was observed in 80% of the samples collected. Dominance of O. oeni was reflected in the bacterial load (O. oeni/total bacteria) measured by qPCR, corresponding to >85% in most of the samples. A total of 178 LAB isolates were identified after sequencing molecular markers, 95 of them corresponded to O. oeni. Further genetic analyses were performed using MLST (7 genes) including 10 commercial strains; the results indicated that commercial strains were grouped together, while autochthonous strains distributed among different genetic clusters. To pre-select some autochthonous O. oeni, these isolates were also characterized based on technological tests such as ethanol tolerance (12 and 15%), SO2 resistance (0 and 80 mg l−1), and pH (3.1 and 3.6) and malic acid transformation (1.5 and 4 g l−1). For comparison purposes, commercial strain VP41 was also tested. Based on their technological performance, only 3 isolates were selected for further examination (genome analysis) and they were able to reduce malic acid concentration, to grow at low pH 3.1, 15% ethanol and 80 mg l−1 SO2. The genome analyses of three selected isolates were examined and compared to PSU-1 and VP41 strains to study their potential contribution to the organoleptic properties of the final product. The presence and homology of genes potentially related to aromatic profile were compared among those strains. The results indicated high conservation of malolactic enzyme (>99%) and the absence of some genes related to odor such as phenolic acid decarboxylase, in autochthonous strains. Genomic analysis also revealed that these strains shared 470 genes with VP41 and PSU-1 and that autochthonous strains harbor an interesting number of unique genes (>21). Altogether these results reveal the presence of local strains distinguishable from commercial strains at the genetic/genomic level and also having genomic traits that enforce their potential use as starter cultures.

A simple molecular method for monitoring co-inoculated yeasts during wine fermentation

The inoculation of Torulaspora into wine is an increasingly popular strategy for improving aromatic profiles. However, questions remain regarding the use of this yeast for alcoholic fermentation (AF). This work presents a simple method based on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of internal transcribed spacers (ITSs) that can be used to distinguish between Torulaspora and Saccharomyces, the most commonly co-inoculated yeasts in wines.