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Wine sample microorganism populations amplification by TruePrime® WGA kit for a Biome analysis with WineSeq technology

 

By Dr Alberto Acedo.

Biome Makers Co founder

http://www.biomemakers.com

Introduction

Biome Makers is a biotech startup specialized in microbiome understanding to provide comprehensive analysis for agricultural and industrial processes.

Winemakers are faced with decisions throughout the winemaking process: from the growth conditions of their wines (choice of land, pruning, irrigation) to the time of harvest, and after harvest (barrel oak, cultured or wild yeast, maceration time, fermentation temperature, micro-oxygenation, etc). All of these decisions alter the contribution of the initial microbial communities to the final product, and thus alter the expression of the terroir. Currently, the choice is between two extremes: either producing wine that is terroir-driven, unique to and dependent on the naturally occurring microbial communities and often less predictable, or predictable winemaking with added selected yeast strains, but that loses the emblematic signature of its terroir.

We´ve developed Wineseq a technology that bridges this gap: we have developed a method to identify the relevant microbial communities throughout the winemaking process, from the soil to the bottle, and the data science to interpret the results. We empower winemakers with the knowledge of the microbial dimension of their vineyards’ terroir enabling them to maximize its potential, shape and craft their individual wines’ properties, and anticipate early problems.

It leverages advances in genomic analysis and translates them to the winemaking space by:

  • Identify the native Micro Wine Makers (MWM) in the soil which make your wine unique.
  • Compare your soil microbiome to other regions
  • Estimate the organoleptic potential of your wine Assess necessity of inoculums and sulfur doses
  • Anticipate contamination due to unwanted microorganisms

The identification of the microbial communities involved in the wine transformation after the bottling process, being this a crucial information for the winemakers understanding of the evolution of final product they launch to the consumers’ judgment.

It´s supposed that bacterial communities could survive after the sulfur treatment and the filtrations performed in the wineries. This would mean the existence of microorganisms, and therefore, the bottle would not be fully sterile. The potential microorganisms still living there, due to the low oxygen concentration, would grow at very slow pace and would influence the organoleptic evolution of the bottle. This evolution requires in some cases several years to reach the optimal point for consume.

We needed a WGA method that allow us to get a balanced amplification of the different potential microbial communities existing in the sample. Any specie can be of interest for analysis and for a biome data proper interpretation. On top of this, avoiding potential contamination it´s a priority for us.

We thought that TruePrime® could be the right choice due to its specific primer-free Whole Genome Amplification approach.

While the current gold standard MDA relies on short oligonucleotides to start off the amplification, TruePrime® is based on a combination of the highly processive Phi29 DNA polymerase with the recently discovered primase/polymerase TthPrimPol. In this setup, TthPrimPol synthesizes the DNA primers needed for Phi29 DNA polymerase.

These are the key parameters that determine the quality of amplification of genomic DNA we looked for:

  • Strongly minimize contaminations and artefacts in the reaction products.
  • Coverage breadth and uniformity.
  • Femto-/attogram range detection
  • Absence of artefacts derived from synthetic oligonucleotides present in random-primed methods.
  • Very low amplification bias

Methodology

Wineseq methodology is based on metagenomic approaches and NGS workflows.

Sample and DNA isolation.

We selected 200 ml of wine “Crianza de Toro” from a bottle bought in a shop in Oakland. The bottle for the wine extraction was open in laminar flow hood under sterile conditions. For the DNA Isolation we used a method by physical disruption using bead beating. The sampling and DNA isolation process was done duplicated.

DNA WGA amplification

We followed the handbook protocol recommendations. We used 2,5 µl of purified gDNA and ran the isothermal amplification reaction during 6 hours.

We obtained 2,5 µg of amplified DNA (50 µl at 50 ng/µl).

Library Preparation and Sequencing

The library preparation was performed with Nextera XT kit (Illumina) following protocol instructions by manufacturer.

Both libraries were sequenced in a Miseq 301×2 (pair end) obtaining a total of 21.982.976 reads, approximately 10M for each library.

Results and Discussion

We obtained 21.982.976 reads by the NGS process. 7,54% of those have been assigned to the bacterial kingdom.

Most of these bacterial sequences (around 70%) belong to Acetobacter pasteurianus. This is one of the most common organisms responsible for spoilage during storage and ageing. They arrive at the winery on grape berry surfaces and continue to persist throughout fermentation although their population numbers are lowered due to the presence of increased amounts of ethanol and lack of oxygen.

Oenococcus oeni was also detected in the samples. This is part of the normal grape and fermentation flora in wine. It is desired for malolactic fermentation at low pH.

Oenococcus oeni and Acetobacter Pasterianus are part of the succession of microbial populations involved in the wine production. Oenococcus oeni is responsible for malolactic fermentation, followed by acetobacter, once the wine is moved to cask. After this point in the process, genders like gluconobacter and gluconoacetobacter.

It´s logic to think about an abundant representation of acetobacter, gluconobacter and gluconoacetobacter due to their presence in processes run in the cask. However, it is unexpected the detection of Oenococcus oeni, the responsible for the malolactic fermentation previous to wine movement to the cask.

Regarding the fungi kingdom, 10% of the reads belongs to Saccharomyces cerevisiae which is the most common yeast associated with winemaking. Although this yeast is not the most abundant species in grapes and fresh must, it dominates the fermentation process due to its robust fermentation capabilities, tolerance of relatively high levels of alcohol and sulfur dioxide as well as its ability to thrive in normal wine pH between 2.8 and 4.

50% of the reads were not assigned to any specie, but this a very common output in a metagenomic approach.

Conclusion

By Using WGA TruePrime® technology by Sygnis we were able to amplify a balanced representation of the microbial populations coexisting in a wine sample from a commercial bottle. This amplification technology was suitable for the downstream NGS workflow within the Wineseq methodology.

Wine is a live entity in which exists a succession of different microbial entities, bacteria and yeasts. These species influence the organoleptic properties of the wine. Some of them overpass the wine production process and end up in the bottle. This fact opens a new channel for further investigations considering wine as a probiotic product. Further developments for more accurate strategies in the wine aging and conservation stages will be carried out within the R&D programme of Biome Makers Inc.

Dr Alberto Acedo.

Biome Makers Co founder

http://www.biomemakers.com