Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing

Summary

Here, we present a protocol to describe amplicon metagenomic for determining the bacterial community of Traminette grapes, fermenting grapes, and final wine.

Abstract

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/µL to 87 ng/µL. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

Introduction

Traminette grape is characterized by production of superior wine quality, in addition to appreciable yield and partial resistance to several fungal infections^1,2,3. The natural fermentation of grapes relies on associated microorganisms, wine production environment, and fermentation vessels^4,5. Oftentimes, many wineries rely on wild yeast and bacteria for fermentation, production of alcohol, esters, aroma, and flavor development⁶.

The goal of this study is to examine ....

Protocol

1. Experimental wine production

1. Obtain Traminnette grapes from Dynamis Estate wine in Jonesville, North Carolina vineyard, destem, and crush to release must into two separate 600 L open-top fermenters and leave on skins for about 4 days with the lids on.
2. Punch the caps down once a day to keep skins wet and limit volatile acid (VA) production.
   NOTE: The idea is that a lot of the aromatic precursors (monoterpenes) are situated in the skins and leave the juice in contact with th.......

Discussion

The protocol of metagenomics starts from the sampling of the grape must, and when yeast was added to the must, the fermenting wine and final wine samples. This was followed by duplicate DNA extraction that was successfully extracted from these samples. The quantities obtained varied in concentration from 15 ng/ µL to 87 ng/ µL. This shows that the DNA extraction protocol is effective for metagenomic studies of wine. Although the quality of the DNA at A260/A280 varies, this may be attributed to different paramet.......

Materials

Name	Company	Catalog Number	Comments
Agarose gel	Promega, Madison, WI USA	V3121	Electrophoresis
FastPrep DNA spinKit for soil	MP Biomedicals, Solon, OH USA	116560-200	DNA extraction
FastQC software	Babraham Institute, United Kingdom		Bioinformatics
Fermaid O	Scott Laboratory, Petaluma, CA USA		Fermentation
High-Fidelity PCR Master Mix	New England Biolabs, USA	F630S	Polymerase chain reaction for sequencing
NEBNext Ultra	New England Biolabs, USA	NEB #E7103	DNA Library Prep
NEBNext Ultra II DNA Library Prep Kit	Illumina, San Diego, CA  USA		DNA sequencing
NovaSeq Control Software (NVCS)	Illumina, San Diego, CA  USA		DNA sequencing
Novaseq6000 platform	Illumina, San Diego, CA  USA		DNA sequencing
QuiBit	Thermoscientific, Waltham, MA, USA		DNA quantification
Quickdrop spectrophotometer	Molecular device, San Jose, CA, USA		DNA quantification
Sodium Phosphate	Sigma Aldrich	342483	DNA extraction buffer
Stimula Sauvignon Blanc	Scott Laboratory, Petaluma, CA USA		Fermentation