IVAS 2022 banner
IVES 9 IVES Conference Series 9 IVAS 9 IVAS 2022 9 Exogeneous C-S lyase enzyme, a potential tool for thiol enhancement in wine or beer?

Exogeneous C-S lyase enzyme, a potential tool for thiol enhancement in wine or beer?

Abstract

Varietal thiols are considered for years as key aroma compounds in many wines. Their main origin is the cleavage during alcoholic fermentation of S-conjugate precursors present in grapes and musts, even if the levels of precursors already identified struggle to completely explain the levels of thiols found in wine.
In this bioconversion process, yeast is the key since the cleavage of thiol precursors has been demonstrated to be due to the internal C-S lyase activity of the yeast strains. Whatever the intrinsic capacity of the yeast and the regulation mechanisms identified for the transportation of thiol precursor into the cell, the conversion yield remains very low, estimated in most cases at 1%. In this context, the use of exogenous C-S lyases could be an alternative option to reveal a larger part of the thiol aroma.
Our study focused on the characterization of a recombinant C-S lyase model obtained by from Lactobaccilus delbrueckii subsp. bulgaricus, expressed in E. coli [1] to investigate the possibility of using such enzymes in different matrices such as wine, beer or directly hops, known to be rich in thiol precursors.
A spectrophotometric method was developed for the quantification of the C-S lyase activity, using commercially available S-4-nitrophenyl-L-cysteine (Cys(4NP)). This method was then used to study the specificity of substrate and potential competitor recognition, still using Cys(4NP) but also G-4NP, Cys(4NP)-Gly and γGlu-Cys(4NP) conjugates, which were specifically synthesized in our lab, based on our previous results [2].
The C-S lyase we used was able to cleave efficiently Cys(4NP), but not glutathione and dipeptide 4NP-S-conjugates. As expected, pH emerged as a key parameter: no reaction at wine pH (2.8-3.5), low activity at beer pH (4.5-5.6) and > 80% of activity at pH above 6. Assays with N-Ac-Cys(4NP) confirmed that a free amine group on the substrate was compulsory for recognition by the enzyme and subsequent cleavage of the substrate. Free cysteine has also been demonstrated to compete with Cys(4NP) resulting in a dramatic decrease in conversion efficiency.
These first results documented the possibility of using such enzyme in the different matrices, highlighting the constraints for the subsequent identification of C-S lyase more suitable to wine or beer productions

References

[1] Allegrini, A.; Astegno, A.; La Verde, V.; Dominici, P. Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavor applications. J. Biochem. 2017, 61, 349−360.
[2] Bonnaffoux, H., Roland, A., Rémond, E., Delpech, S., Schneider, R., & Cavelier, F. (2017). First identification and quantification of S-3-(hexan-1-ol)-γ-glutamyl-cysteine in grape must as a potential thiol precursor, using UPLC-MS/MS analysis and stable isotope dilution assay. Food Chemistry, 237, 877–886.

DOI:

Publication date: June 27, 2022

Issue: IVAS 2022

Type: Poster

Authors

Clerat Luigi1, Vives Eric1, Cavelier Florine2, Remond Emmanuelle2 and Schneider Rémi3

1PhyMedExp – Physiologie & médecine expérimentale du Cœur et des Muscles [U 1046]
2Institut des biomolécules Max Mousseron (IBMM) – UMR-5247 – CNRS
3Oenobrands Montpellier FR

Contact the author

Keywords

C-S lyase, varietal thiol precursors, wine aroma, S-conjugates

Tags

IVAS 2022 | IVES Conference Series

Citation

Related articles…

1H-NMR-based Metabolomics to assess the impact of soil type on the chemical composition of Mediterranean red wines

The aim of this study was to evaluate the effects of different soil types on the chemical composition of Mediterranean red wines, through untargeted and targeted 1H-NMR metabolomics. One milliliter of raw wine was analyzed by means of a Bruker Avance II 400 spectrometer operating at 400.15 MHz. The spectra were recorded by applying the NOESYGPPS1D pulse sequency, to achieve water and ethanol signals suppression. No modification of the pH was performed to avoid any chemical alteration of the matrix. The generation of input variables for untargeted analysis was done via bucketing the spectra. The resulting dataset was preprocessed prior to perform unsupervised PCA, by means of MetaboAnalyst web-based tool suite. The identification of compounds for the targeted analysis was performed by comparison to pure compounds spectra by means of SMA plug-in of MNova 14.2.3 software. The dataset containing the concentrations (%) of identified compounds was subjected to one-way analysis of variance (ANOVA) to highlight significant differences among the wines. The untargeted analysis, carried out through the PCA, revealed a clear differentiation among the wines. The fragments of the spectra contributing mostly to the separation were attributed to flavonoids, aroma compounds and amino acids. The targeted analysis leaded to the identification of 68 compounds, whose concentrations were significant different among the wines. The results were related to soils physical-chemical analysis and showed that: 1) high concentrations of flavan-3-ols and flavonols are correlated with high clay content in soils; 2) high concentrations of anthocyanins, amino acids, and aroma compounds are correlated with neutral and moderately alkaline soil pH; 3) low concentrations of flavonoids and aroma compounds are correlated with high soil organic matter content and acidic pH. The 1H-NMR metabolomic analysis proved to be an excellent tool to discriminate between wines originating from grapes grown on different soil types and revealed that soils in the Mediterranean area exert a strong impact on the chemical composition of the wines.

Revealing the Barossa zone sub-divisions through sensory and chemical analysis of Shiraz wine

The Barossa zone is arguably one of the most well-recognised wine producing regions in Australia and internationally; known mainly for the production of its distinct Shiraz wines. However, within the broad Barossa geographical delimitation, a variation in terroir can be perceived and is expressed as sensorial and chemical profile differences between wines. This study aimed to explore the sub-division classification across the Barossa region using chemical and sensory measurements. Shiraz grapes from 4 different vintages and different vineyards across the Barossa (2018, n = 69; 2019, n = 72; 2020, n = 79; 2021, n = 64) were harvested and made using a standardised small lot winemaking procedure. The analysis involved a sensory descriptive analysis with a highly trained panel and chemical measurement including basic chemistry (e.g. pH, TA, alcohol content, total SO2), phenolic composition, volatile compounds, metals, proline, and polysaccharides. The datasets were combined and analysed through an unsupervised, clustering analysis. Firstly, each vintage was considered separately to investigate any vintage to vintage variation. The datasets were then combined and analysed as a whole. The number of sub-divisions based on the measurements were identified and characterised with their sensory and chemical profile and some consistencies were seen between the vintages. Preliminary analysis of the sensory results showed that in most vintages, two major groups could be identified characterised with one group showing a fruit-forward profile and another displaying savoury and cooked vegetables characters. The exploration of distinct profiles arising from the Barossa wine producing region will provide producers with valuable information about the regional potential of their wine assisting with tools to increase their target market and reputation. This study will also provide a robust and comprehensive basis to determine the distinctive terroir characteristics which exist within the Barossa wine producing region.

Effects of organic mulches on the soil environment and yield of grapevine

Farming management practices aiming at conserving soil moisture have been developed in arid and semiarid-areas facing water scarcity problems. Organic mulching is an effective method to manipulate the crop-growing microclimate increasing crop yield by controlling soil temperature, and retaining soil moisture by reducing soil evaporation. In this sense, the effectiveness of different organic mulching materials (straw mulch and grapevine pruning debris) applied within the row of a vineyard was evaluated on the soil and on the vine in a Tempranillo vineyard located in La Rioja (Spain). Organic mulches were compared with a traditional bare soil management technique (based on the use of herbicides to avoid weed incidence). Mulching coverages favourably influenced the soil water retention throughout all the grapevine vegetative cycle. However, the soil-moisture variation was not the same under different mulching materials, being the straw mulch (SM) the one that retained more water in comparison with grapevine pruning debris (GPD) based-cover. The changes of soil moisture in the upper surface layer (0–10 cm) were highly dynamic, probably due to water vapour fluxes across the soil-atmospheric interface. However, both, SM and GPD reduced these fluctuations as compared with bare soils. A similar trend occurred with soil temperature. Both organic mulches altered soil temperature in comparison with bare soil by reducing soil temperature in summer and raising it in winter. Moreover, the same buffering effect for the temperature on the covered soil also remains in the deeper layers. To conclude, we could see that organic mulching had a positive impact on soil-moisture storage and soil temperature and the extent of this effect depends on the type of mulching materials. These changes led to higher rates of photosynthesis and stomatal conductivity compared to bare soils, also favouring crop growth and grape yields.

How can historical cultivars mitigate the effects of climate change?

IFV, INRAe and the national network “Partenaires de la Sélection Vigne” representing 37 organizations from the different wine regions, have been working increasingly closely over the last 2 decades towards the preservation of the French varietal patrimony. There are approximately 600 patrimonial varieties according to INRAe and SupAgro Montpellier experts, including ancient cultivars (400) and intravarietal crossbreeds obtained since the 19th century. In the context of a drastic reduction in such varieties from the mid 1980’s in favor of mainstream varieties, it was essential to carry out an inventory of old vines and vineyards. INRAe Vassal collection plays a key role here as it holds the largest diversity available, along with a rich bibliography and herbariums, offering us the opportunity to document and double check the identity of a cultivar, consolidating the expertise of ampelographers. The work is carried out in several stages, from verifying the existence of a variety in a small region, through to rehabilitation. During this session, the authors present the process that leads to the official registration of a variety. After this, IFV selection center takes over to initiate the process of selection and propagation. A specific focus within regions such as the Alps, Champagne and the South-West will provide details of the full procedure. Bia, Bouysselet, Chardonnay rose, Mecle and the aptly named Tardif, are some of the cultivars that have followed this procedure. Furthermore, a recent regulation established by INAO on “varieties of interest for adaptation purposes” might boost uptake by growers. Since 2006, 36 historical cultivars have been registered. Most of these have been neglected in the past due to late maturity, lack of sugar and high titratable acidity at harvest time. Such characteristics are today considered as positive qualities, not only in mitigation of the effects of climate change, but also as an opportunity for restoring diversity…

Measurement of redox potential as a new analytical winegrowing tool

Excell laboratory has initiated the development of an analytical method based on electrochemistry to evaluate the ability of wines to undergo or resist to oxidative phenomena. Electrochemistry is a powerful tool to probe reactions involving electron transfers and offers possibility of real-time measurements. In that context, the laboratory has implemented electrochemical analysis to assess oxidation state of different wine matrices but also in order to evaluate oxidative or reduced character of leaf and soil. Initially, our laboratory focused on dosage of compounds involved in responses of plant stresses and we were also interested in microbiological activity of soils. These analyses were compared with the measurement of redox potential (Eh) and pH which are two fundamental variables involved in the modulation of plant metabolism. Indeed, the variation of redox states of the plant reflects its biological activity but also its capacity to absorb nutriments. The Eh-pH conditions mainly determine metabolic processes involved in soil and leaf and our goal is to determine if this combined analytical approach will be sufficiently precise to detect biological evolutions (plant health, parasitic attack…).