Name: Video: DNA Extraction from Fermented Grape for Metagenomic Analysis
Uploaded: 2023-12-01T14:50:00
Description: 64 Views. DNA Extraction from Fermented Grape for Metagenomic Analysis

dna extraction from fermented grape for metagenomic analysis

Profiling the Bacterial Community in Grapes Using Amplicon Metagenomics

Traminette grape is characterized by production of superior wine quality, in addition to appreciable yield and partial resistance to several fungal infections1,2,3. The natural fermentation of grapes relies on associated microorganisms, wine production environment, and fermentation vessels4,5. 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 the bacterial composition of grapes and monitor their dynamics during fermentation. Although, the modern use of starter cultures such as Saccharomyces cerevisiae for primary fermentation, where alcohol is produced, is common to different wine styles7. In addition, secondary fermentation, where malic acid is decarboxylated by Oenococcus oeni to lactic acid, improves the organoleptic and taste profile of the wine and reduces the acidity of wine8,9. With the recent advances in the use of culture-independent methods, it is now possible to determine different microbes associated with the wine grape and the species that are transferred to must and participate in the fermentation at different times up to the final product10.
The roles and dynamics of wild bacteria from different grapes transferred to the must during wine fermentation are poorly understood. The taxonomy of many of these bacteria is not even known, or their phenotypic properties are uncharacterized. This makes their application in coculture fermentation still poorly underutilized. However, microbiological culture-based analysis has been used to determine the bacterial population associated with grapes and wine10. It is widely known that selective culture plating is tedious, prone to contamination, has a low reproducibility, and output can be doubtful; it also misses bacterial species whose growth requirements are unknown. Previous studies indicate that culture-independent, 16S rRNA gene-based methodologies offer a more dependable and cost-effective approach to characterizing complex microbial communities11. For example, sequencing the hypervariable regions of the 16S rRNA gene has been successfully employed to study bacteria in grapes leaves, berries, and wine12,13,14. Studies have shown that the use of either 16S rRNA metabarcoding or whole metagenomic sequencing is suitable for microbiome studies15. There is emerging information about the possible linkage of bacterial diversity to their metabolic attributes during wine production, which could help in the determination of oenological properties and terroir16.
The need to maximize the advantages of the metagenomic tools using next-generation sequencing (NGS) to study the grape and wine microbial ecology has been emphasized16,17. Also, the use of culture-independent methods based on high throughput sequencing to profile microbial diversity of the food and fermentation ecosystem has become very relevant and valuable to many laboratories and is recommended for industrial use18,19. It provides an advantage of detection and taxonomic profiling of the present microbial populations and the contribution of environmental microbes, their relative abundance, and alpha and beta diversity20. The sequencing of the variable region of the 16S region has become an important gene of choice and has been used during different microbial ecological studies.
While many studies focus on fungi, especially yeast, during wine fermentation21, this study reported the 16S amplicon sequencing and bioinformatic tools used to study the bacteria during Traminette grapes fermentation for wine production.

Author Spotlight: Exploring the Fermentation Microbiome Through Next-Generation Sequencing

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

DNA Extraction from Fermented Grape for Metagenomic Analysis

dna-extraction-from-fermented-grape-for-metagenomic-analysis

Research

JoVE Journal

Biology

64 Views. DNA Extraction from Fermented Grape for Metagenomic Analysis

Video: DNA Extraction from Fermented Grape for Metagenomic Analysis

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/&#181;L to 87 ng/&#181;L. 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.

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