Grape and wine microorganisms: diversity and adaptation

Developments in high-throughput sequencing (HTS) technologies allow us to obtain large amounts of microbial information from wine and must samples. Thus approaches, that are aimed at characterising the microbial diversity during fermentation, can be enhanced, or possibly even replaced, with HTS-based metabarcoding. To reduce experimental biases and increase data reproducibility, we compared 3 DNA extraction methods by evaluating differences in the fungal diversity with Riesling alcoholic fermentation samples at four different vineyards. The fungal diversity profiling was done using the genetic markers ITS2 and D2 using metabarcoding. The extraction methods compared consisted of a commercial kit, a recently published protocol that includes a DNA enhancer, and a protocol based on a buffer containing common inhibitor removal reagents. All methods were able to distinguish vineyard effects on the fungal diversity, but the results differed quantitatively.

Sulfur dioxide (SO2) is used in winemaking due of its antioxidant, antioxydasic and antiseptic properties. Excessive amount of SO2 can negatively impact wine sensory perception and be detrimental for health. Agri-food industries are more transparent towards consumers concerning addition of sulfites, and oenology is no exception in this clairvoyance. As a consequence, the increase of consumers preference for wine with low or absent of sulfites addition is notorious. In this context, the impact of low/zero sulfites winemaking process on the microbial community should be evaluated. Moreover, microbial agents corresponding to bioprotective cultures represent a growing interest as an alternative to sulfites preservation in the early stages of vinification. However, scientific studies conducted to demonstrate their real effect are almost rare.

Extracellular substances (ECS) represent all molecules outside the cytoplasmic membrane, which are not directly anchored to the cell wall of microorganisms living through a planktonic or biofilm phenotype. They are the high-biomolecular-weight secretions from microorganisms (i.e. extracellular polymeric substances – EPS – proteins, polysaccharides, humic acid, nucleic acid), and the products of cellular lysis and hydrolysis of macromolecules. In addition, some high- and low-molecular-weight organic and inorganic matters from environment can also be adsorbed to the EPS. All can be firmly bound to the cell surface, associated with the EPS matrix of biofilm, or released as being freely diffusing throughout the medium.

Wine biological aging is characterized by the development of yeast strains that form a biofilm on the wine surface after alcoholic fermentation. These yeasts, known as flor yeasts, form a velum that protects the wine from oxidation during aging. Thirty-nine velums aged from 1 to 6 years were sampled from “Vin jaune” from two different cellars. We show for the first time that these velums possess various aspects in term of color and surface aspects. Surprisingly, the heterogeneous velums are mostly composed of one species, S. cerevisiae. Scanning electron microscope observations of these velums revealed unprecedented biofilm structures and various yeast morphologies formed by the sole S. cerevisiae species.

Through the years, extensive studies have been conducted on fungal biodiversity during the winemaking process: from the vineyard until aging.

SO2 is used during winemaking for its anti-oxidative and anti-microbial properties. A high SO2 concentration in the wine has negative impacts by hiding wine aromas and delaying malolactic fermentation.

Nowadays the use of non-Saccharomyces as starters of alcoholic fermentation (AF) has increased because of the modulation of the organoleptic profile of wines

Alcoholic fermentation is the main step for winemaking, mainly performed by the yeast Saccharomyces cerevisiae. But other wine yeasts called non-Saccharomyces may contribute to alcoholic fermentation and modulate the wine aroma complexity. The recurrent problem with the use of these non-Saccharomyces yeasts is their trend to die off prematurely during alcoholic fermentation, leading to a lack of their interesting aromatic properties searched in the desired wine. This phenomenon appears to be mainly due to interactions with S. cerevisiae. These interactions are often negatives but remain unclear because of the species and strain specific response. Among the non-Saccharomyces yeasts, Lachancea thermotolerans is a wine yeast naturally found in grape must and well known as a great L-lactic acid producer and an aromatic molecules enhancer, but its behavior during alcoholic fermentation can be completely different in co-fermentation with S. cerevisiae in function of strain used.

Over the last years, the potential use of non-Saccharomyces yeasts to modulate the production of target metabolites of oenological interest has been well recognized. Among non-Saccharomyces yeasts, Starmerella bacillaris (synonym Candida zemplinina) is considered one of the most promising species to satisfy modern market and consumers preferences due to its peculiar characteristic (enhance glycerol and total acidity contents and reduce ethanol production). Mixed fermentations using Starm. bacillaris and Saccharomyces cerevisiae starter cultures represent a way to modulate metabolites of enological interest, taking advantage of the phenotypic specificities of the former and the ability of the latter to complete the alcoholic fermentation. However, the consumption of nutrients by these species and their produced metabolites may inhibit or stimulate the growth (and malolactic activity) of lactic acid bacteria (LAB).

Nowadays the use of Torulaspora delbrueckii is more and more common in winemaking. However, its behavior in presence of Saccharomyces cerevisiae is not always predictable.

The elaboration of sparkling wine is a demanding process requiring technical as well as scientific skills. Uncovering the role of the terroir to the final product quality is of great importance for the wine market. Although the impact of the yeast strains and their metabolites on the final product quality is well documented, the action of bacteria still remains unknown. The malolactic fermentation (MLF) is carried out by the lactic acid bacteria after the alcoholic fermentation in order to ensure the microbial stability during the second fermentation that takes place in the bottle or in tanks. Oenococcus oeni is the only selected species to drive MLF that has been commercialized for sparkling wine elaboration and it is naturally present on grapes, in the cellar and also in the final product. However, whether the bacterial strain contributes to the sensory characteristics of sparkling wine is still questioned.

In winemaking, several antimicrobial peptides (AMPs) produced by different strains of Saccharomyces cerevisiae were found to be responsible for the inhibition of malolactic fermentation (MLF) carried out by some strains of Oenococcus oeni. However, only two AMPs produced by one of the yeast strains studied were totally identified and their mechanism of action was described. In an attempt to identify new AMPs, a 5-10 kDa peptidic fraction produced by an oenological strain of S. cerevisiae and previously shown to strongly inhibit MLF carried out by a strain of O. oeni was further purified.

During wine alcoholic fermentation, yeasts produce volatile aroma compounds from sugar and nitrogen metabolism. Some of the metabolic pathways leading to these compounds have been known for more than a century.

Over the last decade, the use of non-Saccharomyces yeasts in the winemaking process has been re-assessed and accepted by winemakers. These yeasts can be used to achieve specific objectives such as lowering the ethanol content, preventing wine spoilage and increasing the production of specific aroma compounds. Since these species are unable to complete alcoholic fermentation, strategies of co- and sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae have been developed. However, when mixed starter cultures are used, several parameters (e.g. strain yeast, inoculation timing and nutrient competitions) impact the growth of the individual yeasts, the fermentation kinetics and the metabolites/aroma production. In particular, competition for nitrogen compounds could have a major impact, potentially leading to sluggish fermentation when the yeast assimilable nitrogen (YAN) availability is low. Moreover, many aroma compounds produced by the yeasts are directly produced and influenced by nitrogen metabolism such as higher alcohols, acetate esters and ethyl esters which participate in the organoleptic complexity of wine.

Grapes from warm(ing) climates often contain excessive sugars but lack acidity. This can lead to highly alcoholic wines with compromised stability and balance. The yeast Lachancea thermotolerans can ameliorate such wines due to its metabolic peculiarity – partial fermentation of sugars to lactic acid. This study aimed to elucidate the population-wide diversity in L. thermotolerans, whilst selecting superior strains for wine sector. An extensive collection of isolates (~200) sourced from different habitats worldwide was first genotyped on 14 microsatellite loci. This revealed differentiation of L. thermotolerans genetic groups based on the isolation substrate and geography. The 94 genotyped strains were then characterised in Vitis vinifera cv. Chardonnay fermentations.