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…

Spatial variability of temperature is linked to grape composition variability in the Saint-Emilion winegrowing area

Elevated temperature during the grape maturation period is a major threat for grape quality and thus wine quality. Therefore, characterizing the grape composition response to temperature at a larger scale would represent a crucial step towards adaptation to climate change. In response to changes in temperature, various physiological mechanisms regulate grape composition. Primary and secondary metabolisms are both involved in this response, with well-known effects, for example on anthocyanins, and lesser known effects, for example on aromas or aroma precursors. At the field scale or at the regional scale, however, numerous environmental or plant-specific factors intervene to make the effects of temperature difficult to distinguish from overall variability. In this study, it was attempted to overcome this difficulty by selecting well-characterized situations with differing temperatures. A long-term study of air temperature variability across several Merlot vineyards in the Saint-Emilion and Pomerol wine producing area found significant temperature differences and gradients at various time scales linked to environmental factors. From this study area, a few sites were selected with similar age, soil and training system conditions, and with repeated and contrasted temperature differences during the maturation period. The average temperature difference during the maturation period was about 2°C between cooler and warmer sites, a difference similar to that expected under future climate change scenarios. In close vicinity to the temperature sensors at each site, grape berries were sampled at different times until full maturity during 2019 and 2020. Also, berries from bunches on either side of the row were analyzed separately, allowing an investigation of bunch exposure effect associated with the coupling of berry temperature and solar radiation. Four replicates of pooled berries for each time – site – bunch exposure combination were obtained and analyzed for biochemical composition. Analyses of variance of the biochemical composition data collected at different sampling times reveal significant effects associated with temperature, site, and bunch azimuth. For instance, anthocyanins in grape skins are clearly influenced by temperature and solar radiation exposure, with up to 30% reduction in warmer conditions.

Combining effect of leaf removal and natural shading on grape ripening under two irrigation strategies in Manto negro (Vitis vinifera L.)

The increasingly frequent heat waves during grape ripening pose challenges for high quality wine grape production. Defoliation is a common practice that can improve the control of diseases in bunches, but also it increases the exposure to sunlight. Grapes exposed to solar radiation reach temperatures over the optimum for berry development and maturation. This makes the development of irrigation and canopy management techniques of great importance to maximize yield and grape quality. A field experiment was carried out during 2021 using Manto negro wine grapes to study the effect of applied irrigation and different light exposure levels on grape quality. Two irrigation treatments were imposed based on the frequency and amount of water doses in a four-block experimental vineyard at Bodega Ribas (Mallorca). Three light exposure treatments were randomly applied in each irrigation plot. The light treatments included exposed clusters from pea size, non-exposed clusters, and shaded clusters after softening. Leaf area index and canopy porosity was estimated every 2 weeks. Midday leaf water potential was measured weekly. Additionally, apparent electrical conductivity was measured between rows to estimate the soil water content variability. Light and temperature sensors were installed at the bunch level to quantify the differences in bunch temperature and light intensity among treatments. The effect of irrigation and cluster light exposure on berry weight, TSS, TA, malic acid, tartaric acid, K+, and pH were analysed at 5 moments along grape ripening. During different heat waves, the natural shading technique decreased the maximum bunch temperature around 10 °C respect to the exposed bunches in both irrigation strategies. The combination of defoliation and shading techniques after softening decreased TSS at harvest and affected most of the quality parameters during the last stages of ripening, showing an interesting technique to delay ripening in warm viticulture areas.

First step in the preparation of a soil map of the Protected Designation of Origin Valdepeñas (Central, Spain)

This work is a first step to make a map of vineyard soils. The characterization of the soils of the Protected Designation of Origin (D.P.O.) Valdepeñas will allow to group the studied profiles according to their physico-chemical characteristics and the concentrations of most relevant chemical elements. 90 soil profiles were analysed throughout the territory and the soils were sampled and described according to FAO (2006) and classified according to and Soil Taxonomy (2014). All samples were air dried, sieved and some physico-chemical parameters were determined following standard protocols. Also, major and trace elements were analysed by X-ray fluorescence. The statistically study was made using the SPSS program. Trend maps were made using the ArcGIS program. The studied soils have the following average properties: pH, 8.3; electrical conductivity, 0,20 dS/m (low); clay, 18.8% (medium) and CaCO3, 17.1% (high). In the study for the major elements. The major elements of these soils are Si, followed by Ca and Al, with an average content of 203.7 g/kg, 105.5 g/kg and 74.0 g/kg respectively. On the other hand, 27 trace elements have been studied. Of all of them, it can be highlighted the average values of Ba (361.8 mg/kg), Sr (129.3 mg/kg), Rb (83.4 mg/kg), V (74.2 mg/kg) and Ce (70.6 mg/kg). Ba, V and Ce values are higher and the values of Sr and Rb are lower to those found in the literature. The discriminant analysis shows a percentage of grouping of 91%. The content of chemical elements together with the physico-chemical characteristics allows grouping the soils in 4 group according to their order in the classification to Soil Taxonomy; due to the importance of the Calcisols in Castilla-La Mancha, it has been decided to establish them as their own group even if they do not appear in Soil Taxonomy classification.

Grape must quality and mesoclimatic variability in Fruška Gora wine-growing region, Serbia

The Fruška Gora mountain is a traditional wine-growing region in Serbia situated in the Pannonian Basin. Due to such a position, the vicinity of the Danube River and the presence of concave configuration, it is suitable for grape production. This paper provides analyses of spatial variations in meteorological parameters and grape juice quality within Fruška Gora wine region over three consecutive vintages (2018-2020). The examined period can be defined as warm with cool nights during September (AVG 18,9°C; GDD 1918°C; CI 12°CF) and with the presence of mesoclimatic variability. The East part of the study area was somewhat drier and hotter compared to other parts of the region. The analyses of grape must samples (190 in total) of five cultivars (Cabernet-Sauvignon, Merlot, Chardonnay, Sauvignon blanc and Grašac (Welschriesling)) commonly grown across the region (19 sites), were performed using Fourier Transform Infrared Technology (FTIR). Among all cultivars, Sauvignon blanc was harvested first in the East area (DOY=246±5, GDD at harvest=1552±74, 22.2±0.7 °Brix), while the latest harvest was recorded for Cabernet-Sauvignon in the West (DOY=283±5, GDD at harvest=1936±187, 23.4±1.0 °Brix ). Both the red and white cultivars had higher acidity and YAN in the grape must if the vines were grown in the North and East compared to South and West areas. According to PCA analysis, Grašac showed the lowest variation in grape must chemical composition. Thus, the results confirm that Grašac is the most stable cultivar in Fruška Gora. All monitored cultivars reached technological fruit ripeness by the end of the growing season. However, it was difficult to reach full ripeness of red cultivars, mostly beacuse of uncoupling of technolocical and phenolic ripeness. Thus, Cabernet-Sauvignon had higher variations in GDD sums at harvest compared to other cultivars, which probably increased variations in grape must quality.

The impact of sustainable management regimes on amino acid profiles in grape juice, grape skin flavonoids, and hydroxycinnamic acids

One of the biggest challenges of agriculture today is maintaining food safety and food quality while providing ecosystem services such as biodiversity conservation, pest and disease control, ensuring water quality and supply, and climate regulation. Organic farming was shown to promote biodiversity and carbon sequestration, and is therefore seen as one possibility of environmentally friendly production. Consumers expect organically grown crops to be free from chemical pesticides and mineral fertilizers and often presume that the quality of organically grown crops is different or higher compared to conventionally grown crops. Integrated, organic, and biodynamic viticulture were compared in a replicated field trial in Geisenheim, Germany (Vitis vinifera L. cv. Riesling). Amino acid profiles in juice, grape skin flavonoids, and hydroxycinnamic acids were monitored over three consecutive seasons beginning 7 years after conversion to organic and biodynamic viticulture, respectively. In addition, parameters such as soil nutrient status, yield, vigor, canopy temperature, and water stress were monitored to draw conclusions on reasons for the observed changes. Results revealed that the different sustainable management regimes highly differed in their amino acid profiles in juice and also in their skin flavonol content, whereas differences in the flavanol and hydroxycinnamic acid content were less pronounced. It is very likely that differences in nutrient status and yield determined amino acid profiles in juice, although all three systems showed similar amounts of mineralized nitrogen in the soil. Canopy structure and temperature in the bunch zone did not differ among treatments and therefore cannot account for the observed differences in favonols. A different light exposure of the bunches in the respective systems due to differences in vigor together with differences in berry size and a different water status of the vines might rather be responsible for the increase in flavonol content under organic and biodynamic viticulture.