Antioxidant inactivated yeast: High potential of non-Saccharomyces specific metabolome.

Understanding the contribution of new natural sources of antioxidant compounds to the stability of wines is of great interest in a context of reduction of sulfites. Here, we investigated the antioxidant potential of selected inactivated non-Saccharomyces yeast (INSY) along with related chemical fingerprints, using combined untargeted UHPLC-Q-ToF MS and DPPH analyses. 4 INSY species were compared to a reference inactivated Saccharomyces cerevisiae yeast (ISY) selected for its high antioxidant capacity. Our results show that, all the INSY can accumulate GSH during the specific production process with yields ranging from +170 % to +360 % compared to the corresponding classical production process. The principal component analysis of the 3511 ions detected by UHPLC-Q-ToF-MS clearly grouped INSY by species, independently of the production process. One INSY exhibited equivalent antioxidant capacity to the control ISY, but with a GSH concentration four times lower (4.73 ± 0.09 mg/g against 20.95 ± 0.34 mg/g, respectively). 73 specific ions presenting strong and significant spearman correlation (rho < -0.6, p-value < 0.05) with the DPPH scores, clustered the most antioxidant INSY and the control Saccharomyces in different groups, indicating that the antioxidant capacity of these two products should be driven by different pools of compounds. These results point out that, GSH alone is not relevant to explain the antioxidant capacity of INSY soluble fractions and other more reactive compounds must be considered, which opens an avenue for the selection new species with great enological potential.

Exogenous C-S Lyase Enzyme, a Potential Tool To Release Aromas in Wine or Beer?

Varietal thiols have an impact on the overall aroma of many white, rosé, and red wines and beers. They originate from the metabolism of non-odorant aroma precursors by yeast during the fermentation step, via an intrinsic enzyme, the carbon-sulfur ß-lyase (CSL, EC 4.4.1.13). However, this metabolism is directly dependent upon efficient internalization of aroma precursors and intracellular CSL activity. Consequently, the overall CSL activity converts on average only 1% of the total precursors available. To improve the conversion of thiol precursors during winemaking or brewing, we investigated the possibility of using an exogenous CSL enzyme from Lactobacillus delbrueckii subsp. bulgaricus produced in Escherichia coli. We first implemented a reliable spectrophotometric method to monitor its activity on different related aroma precursors and studied its activity in the presence of various competing analogues and at different pH values. This study allowed us to highlight the parameters to define CSL activity and structural insights for the recognition of the substrate, which pave the way for the use of exogenous CSL for the release of aromas in beer and wine.

Colorectal cancer testing rates after implementation of an organised screening programme in Vaud, Switzerland.

AIMS OF THE STUDY: Canton Vaud, Switzerland, implemented an organised colorectal cancer  screening programme with colonoscopy and faecal occult blood tests in 2015, 4 to 6 years ahead of neighbouring cantons. Before its implementation, nearly half of Swiss citizens were already up to date with screening, primarily from opportunistic colonoscopies. We hypothesised that earlier implementation of an organised programme would be associated with greater increases in colorectal cancer testing rates. METHODS: We analysed Swiss health insurance claim data from CSS, a Swiss health insurer covering 16% of the Swiss population and 10% of canton Vaud. We stratified 50-69-year-olds into groups from Vaud, its four neighbouring cantons (Fribourg, Geneva, Neuchâtel and Valais), and the rest of Switzerland. We analysed overall, faecal occult blood test and colonoscopy testing rates for each year between 2010 and 2018. RESULTS: The overall testing rate increased from 7.6% in 2010 to 11.6% in 2018 (+4.0%) in Vaud, from 6.1% to 9.3% (+3.2%) in neighbouring cantons and from 7.4% to 8.6% (+1.2%) in the rest of Switzerland. The faecal occult blood test rate increased between 2016 and 2018 from 2.9% to 4.1% (+1.2%) in Vaud and from 1.7% to 2.6% (+0.9%) in neighbouring cantons, but it decreased from 3.1% to 1.5% (-1.6%) in the rest of Switzerland. The colonoscopy rate increased in all cantons, from 4.7% to 7.5% in Vaud (+2.8%), from 4.4% to 6.7% in neighbouring cantons (+2.3%) and from 4.3% to 7.1% in the rest of Switzerland (+2.8%). By 2018, 40% of faecal occult blood tests and 26% of colonoscopies in Vaud occurred in the organised programme. Those who completed an faecal occult blood test within the Vaud programme were younger, had fewer comorbidities and were more likely to have a high-deductible health plan than those tested outside the programme. CONCLUSIONS: Colorectal cancer testing rates increased between 2010 and 2018, with greater absolute increases in Vaud than in neighbouring cantons or the rest of Switzerland. Faecal occult blood test use increased in both Vaud and neighbouring cantons, possibly reflecting changes in testing patterns by general practitioners. By 2018, 40% of colonoscopies and 26% of faecal occult blood tests occurred within the screening programme.

Ten-year changes in colorectal cancer screening in Switzerland: The Swiss Health Interview Survey 2007, 2012 and 2017.

Recent recommendations for colorectal cancer (CRC) screening suggest fecal occult blood test (FOBT) or colonoscopy. Since 2013, mandatory health insurance in Switzerland reimburse CRC screening. We set out to determine if CRC testing rate and type of CRC screening changed in Switzerland from 2007 to 2017 and between the three main language regions. We extracted data on 50-75-year-olds from the Swiss Health Interview Survey (SHIS) 2007, 2012 and 2017 to determine rates of self-reported testing with FOBT within last 2 years and colonoscopy within last 10 years. We estimated prevalence ratio (PR) in multivariate-adjusted logistic regression models and compared rates in German-, French- and Italian-speaking regions, adjusting for sociodemographic, self-rated health and insurance variables. Overall testing rates (FOBT or colonoscopy) increased in all regions from 2007 to 2017 (German-speaking 33.6% to 48.3%; French-speaking 30.8% to 48.8%; Italian-speaking 37.9% to 46.8%), mainly because of an increase in colonoscopy rate for screening reasons (p < 0.001 in all regions). Rates of FOBT testing fell significantly in the German-speaking region (11.9% to 4.4%, p < 0.001), but not in the Italian- (13.9% to 8.5%, p = 0.052) and French-speaking regions (7.6% to 7.4%, p = 0.138). Overall CRC testing rate rose from 33.2% in 2007 to 48.4% in 2017, mainly because of an increase of colonoscopy rate for screening reasons. Coverage remains below the 65% target of European guidelines. Organized screening programs encouraging FOBT screening could contribute to further increasing the CRC testing rate.

Change in colorectal cancer (CRC) testing rates associated with the introduction of the first organized screening program in canton Uri, Switzerland: Evidence from insurance claims data analyses from 2010 to 2018.

The first canton in Switzerland to implement an organized colorectal cancer screening program (OSP) was Uri. Starting in 2013, it offered 50-69-year-olds free testing with colonoscopy every 10 years or fecal occult blood test (FOBT) every 2 years. We tested the association between the OSP and testing rates over time. We analyzed claims data of 50-69-year-olds from Uri and neighboring cantons (NB) provided by a large health insurance and complemented it with data from the OSP. We fitted multivariate adjusted logistic regression models to compare overall testing rates and by method (colonoscopy or FOBT/both) We computed the 2018 rate of the population up-to-date with testing (colonoscopy within 9 years/FOBT within 2 years). Yearly overall testing rates in Uri increased from 8.7% in 2010 to 10.8% in 2018 and from 6.5% to 7.9% in NB. In Uri, the proportion tested with FOBT/both increased from 4.7% to 6.0% but decreased from 2.8% to 1.1% in NB. Testing by FOBT/both increased more between 2015 and 2018 than 2010-2012 in Uri than in NB (OR:2.1[95%CI:1.8-2.4]), it increased less for colonoscopy (OR:0.60[95%CI:0.51-0.70]), with no change in overall CRC testing (OR:0.91[95%CI:0.81-1.02]). In 2018 in Uri, 42.5% were up-to-date with testing (FOBT/both:9.2%, colonoscopy:35.7%); in NBs, 40.7% (FOBT/both:2.7%, colonoscopy:39%). Yearly FOBT rates in Uri were always higher than in NB. Though the OSP in Uri was not associated with a greater increase in overall testing rates, the OSP was associated with increased FOBT.

Change in Colorectal Cancer Tests Submitted for Reimbursement in Switzerland 2012-2018: Evidence from Claims Data of a Large Insurance.

Objectives: Guidelines recommend colorectal cancer (CRC) screening by fecal occult blood test (FOBT) or colonoscopy. In 2013, Switzerland introduced reimbursement of CRC screening by mandatory health insurance for 50-69-years-olds, after they met their deductible. We hypothesized that the 2013 reimbursement policy increased testing rate. Methods: In claims data from a Swiss insurance, we determined yearly CRC testing rate among 50-75-year-olds (2012-2018) and the association with socio-demographic, insurance-, and health-related covariates with multivariate-adjusted logistic regression models. We tested for interaction of age (50-69/70-75) on testing rate over time. Results: Among insurees (2012:355'683; 2018:348'526), yearly CRC testing rate increased from 2012 to 2018 (overall: 8.1-9.9%; colonoscopy: 5.0-7.6%; FOBT: 3.1-2.3%). Odds ratio (OR) were higher for 70-75-year-olds (2012: 1.16, 95%CI 1.13-1.20; 2018: 1.05, 95%CI 1.02-1.08). Deductible interacted with changes in testing rate over time (p < 0.001). The increase in testing rate was proportionally higher among 50-69-years-olds than 70-75-year-olds over the years. Conclusions: CRC testing rate in Switzerland increased from 2012 to 2018, particularly among 50-69-years-olds, the target population of the 2013 law. Future studies should explore the effect of encouraging FOBT or waiving deductible.

The Impact of Hanseniaspora vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), ß-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

The reduction of plant sink/source does not systematically improve the metabolic composition of Vitis vinifera white fruit.

The quality of wine grapes depends on the balance between primary and secondary metabolites. Unlike many perennial crops that accumulate starch in the fruits before ripening, the non-climacteric grapes ripe with no previous carbon reserves. Based on the assumption that fruit carbon sink is limiting metabolite accumulation in grapes, bunch thinning is performed to limit plant Sink/Source (S/S). We studied the effects of severe bunch thinning on the accumulation of primary metabolites and on four families of glycosylated aroma precursors (GAPs) at the arrest of fruit phloem unloading of two white grape Vitis vinifera cvs. At plant level, crop reduction resulted in significant losses of metabolites to be accumulated in the fruits: i.e. up to 72% for sugars, 75% for organic acids and GAPs. Nevertheless, S/S manipulation could not modify the balance between GAPs and primary metabolites or increase the concentration in GAPs in the physiologically ripe grape.

Spotlight on release mechanisms of volatile thiols in beverages.

Volatile thiols are very strong-smelling molecules that can impact the aroma of numerous beverages. Several thiols and thiol precursors have been reported previously in different plants used as raw material for beverages, some of which are fermented. We focused on thiols in beverages and their release mechanisms from precursors during processing. Volatile thiols in beverages can be classified aslow molecular weight volatile thiols (e.g. H2S, methanethiol) which impact the smell negatively, and volatile thiols with higher boiling points that contribute positively to the aroma profile. The first part of this review is devoted to volatile thiols, without considering small malodorous molecules. The second part deals with thiol precursors and the different release mechanisms induced by processing (e.g. extraction, roasting or fermentation) and by the growing methods (e.g. viticulture), which can impact on amounts of thiols and their precursors.

Impact of agronomic practices on grape aroma composition: a review.

Aroma compounds are secondary metabolites that play a key role in grape quality for enological purposes. Terpenes, C13 -norisoprenoids, phenols, and non-terpenic alcohols are the most important aroma compounds in grapes and they can be found as free volatiles or glycoconjugated (bound) molecules. The non-volatile glycosylated group is the largest, and it is present in all varieties of Vitis vinifera (L.), the most widely used species for wine production. These aroma precursors represent the reserve of aroma molecules that can be released during winemaking. Their relative and absolute concentrations at fruit ripening determine the organoleptic value of the final product. A large range of biotic and abiotic factors can influence their biosynthesis in several ways. Agronomic practices such as irrigation, training systems, leaf removal, and bunch thinning can have an effect at plant level. The spraying of stimulatory compounds on fruit at different developmental stages has also been shown to modify metabolic pathways at fruit level with some impact on the aroma composition of the grapevine fruit. Viticulturists could act to promote aroma precursors to improve the aromatic profile of grapes and the wine ultimately produced. However, agronomic practices do not always have uniform results. The metabolic and physiological changes resulting from agronomic practices are unknown because there has not been sufficient research to date. This review presents the state of the art regarding the influences of vineyard agronomic management on the biosynthesis of grape aroma compounds. Although literature regarding the topic is abundant there are still many unknown biological mechanisms involved and/or that have been insufficiently studied. The aim of this work is therefore to find the gaps in scientific literature so that future investigations can focus on them. © 2018 Society of Chemical Industry.

Revisiting the evaluation strategy of varietal thiol biogenesis.

The varietal thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-mercapto-4-methylpentan-2-one (4MMP) are key aroma compounds in wine due to the tropical notes they impart. They are released by yeast during alcoholic fermentation from different precursors. However, a large part of 3MH origin remains unknown. In this study, we focused on dipeptide forms arising from glutathione S-conjugates to 3MH and 4MMP. Using labelled tracers, we showed in spiked must the release of varietal thiols from 4 different compounds. We highlighted the interconversion between different forms of precursors under defined enological conditions. Cysteinyl-glycine S-conjugates are partially degraded into cysteine S-conjugates, contrary to γ-glutamyl-cysteine S-conjugates. Glutathione S-conjugate to 3MH can be partially degraded to γ-glutamyl-cysteine S-conjugate to 3MH. For the first time, all these labeled forms of precursors were found to release 3MH or 4MMP between 0.17 and 1% molar conversion yield. Two different yeasts were compared without any significant difference.

The Microvine, a plant model to study the effect of vine-shoot extract on the accumulation of glycosylated aroma precursors in grapes.

BACKGROUND: The Microvine plant model displays unique reproductive organ behavior and is suitable for grapevine fruit physiological studies, allowing one to undertake studies up to five times more rapidly than the current situation with grapevines. Recently, vine-shoot aqueous extracts, which have an interesting phenolic and aroma composition, have been proposed as viticultural biostimulants, since their post-veraison foliar application to grapevines impacts the wine aroma profile. Using Microvines, the aim of this study was to determine the effect of vine-shoot extract foliar application on 21 stages of grape development. The application was carried out from BBCH 53 (inflorescences clearly visible) to BBCH 85 (softening of berries) to reveal stage-specific responses of the accumulation of glycosylated aroma precursors at BBCH 89 (berries ripe for harvest), the phenological stage selected to study the treatment effect. RESULTS: Microvine use made it possible to carry out 15 sampling time points during 86 days of the experiment, which were established by the cumulative degree days (CDD) parameter. The results confirmed that vine-shoot extract treatment had a positive impact on total glycosylated compounds, especially aglycones such as alcohols, terpenes and C13 -norisoprenoids, with a higher effect when the treatment was applied during ripening. CONCLUSION: Extrapolation of the results to grapevines suggests that vine-shoot extract treatment could modulate the synthesis of grape glycosylated aroma precursors. © 2017 Society of Chemical Industry.

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.

Varietal thiols are key aroma compounds in wine issued from multiple and complex origins. Several precursor families have been identified in grapes and must and have been widely studied. But a large part of thiol origin still remains unknown. Thus, we only have an incomplete picture of thiol precursors and there is a lack of knowledge on pre-fermentative mechanisms that can impact their levels. Our study focused on the formal identification and the quantification of new varietal thiol precursors in must. First of all, we synthesized natural and labeled standards using an original multi-step strategy, then we developed and validated a UPLC-MS/MS method that allowed us to identify and quantify for the first time a dipeptide S-conjugate to 3MH, the γGluCys-3MH, in Sauvignon B. We observed the S-4-mercapto-4-methylpentan-2-one-l-cysteinyl-glycine (CysGly-4MMP) and S-4-mercapto-4-methylpentan-2-one-N-(l-γ-glutamyl)-l-cysteine (γGluCys-4MMP) but at too low concentration to be quantified.

Innovative analysis of 3-mercaptohexan-1-ol, 3-mercaptohexylacetate and their corresponding disulfides in wine by stable isotope dilution assay and nano-liquid chromatography tandem mass spectrometry.

Both 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) were largely studied for the last 20 years due to their pleasant olfactory notes conferred to wine. Until now, many analytical methods focused only on the free forms of both 3MH and 3MHA in wine that provided partial information in the wine aroma evolution. Our study proposes new analytical measurements which allow quantification of both free and disulfide forms of 3MH and 3MHA to better understand the redox phenomenon occurring in wines and further, to orientate wine aroma evolution. Free thiols were analyzed by an original method based on maleimide derivatization allowing in-situ disulfide reduction followed by SIDA-LC-MS/MS analyses exhibiting excellent performances. Indeed, the accuracy ranged from 95 to 110% in three different wine matrices and the repeatability and intermediate reproducibility were inferior to 15% (RSD measurements). Our method exhibited very low limits of detection, which are below to 0.5ng/L and inferior to the perception thresholds of both compounds. Then, this method was applied to three different wines exposed to several oxidative conditions. On the one hand, it was demonstrated that copper sulfate treatment firstly destroyed the total amount of free 3MH to the benefit of thioether and disulfides compounds, with proportions that could be slightly modified by glutathione addition. On the other hand, oxygenation of wines resulted in partial free 3MH destruction to the benefit of thioether compounds. We proposed for the first time an innovative analysis that gives a complete picture of wine aroma, which can be really useful to winemakers to manage wine aroma evolution and to take advantage of the disulfide reservoir.

Development and validation of a high-throughput analysis of glutathione in grapes, musts and wines by Stable Isotope Dilution Assay and LC-MS/MS.

For the first time, we proposed a high-throughput method to quantify glutathione in grapes, musts and wines for all grape varieties using Stable Isotope Dilution Assay (SIDA). Indeed, the use of SIDA as a quantification method is essential to overcome the chemical instability of glutathione. In practice, glutathione was derivatized in-situ with N-ethylmaleimide to block the cysteine residue and to enhance its lipophilic properties. After quenching with acetic acid, samples were directly analyzed by LC-MS/MS (run of 13 min) in Multiple Reaction Monitoring mode using labeled glutathione as internal standard. The validation according to the International Organization of Vine and Wine recommendations demonstrated the high sensitivity (LOD=45 µg L(-1)), accuracy (recovery=112%) and intermediate reproducibility (RSD=12%) of the method. This high-throughput method that requires only 1 mL of matrix, allowed us to analyze 70 samples per day for a moderate cost.

Development of a routine analysis of 4-mercapto-4-methylpentan-2-one in wine by stable isotope dilution assay and mass tandem spectrometry.

The 4-mercapto-4-methylpentan-2-one (4MMP) is a key aroma compound in wines, especially in Sauvignon Blanc ones. Its accurate quantification is quite difficult due to its traces levels and its reactivity in wine conferred by the thiol function. In this paper, we proposed a new method for its quantification in wine without any sample preparation, based on automated derivatization procedure by methoximation and SIDA-SPME-GC-MS/MS analysis. The derivatization procedure was adapted from a previously published method in order to decrease the amount of reagents and the volume of wine (only 3mL are required). The use of SPME and the detection conditions have also been optimized to reach the best sensitivity as possible. The method was then validated according to the International Organization of Vine and Wine recommendations and exhibited excellent performances. Indeed, this method allowed us to quantify the 4MMP in wine at traces levels (LOD=0.19 ng L(-1)) with reproducible results (RSD<15%) and a very good accuracy (recovery=102%).

Analysis of ochratoxin A in grapes, musts and wines by LC-MS/MS: first comparison of stable isotope dilution assay and diastereomeric dilution assay methods.

Ochratoxin A (OTA) exhibits potent nephrotoxic, carcinogenic and teratogenic effects and its maximum level in wines has been set to 2 µg L(-1) by regulation. Consequently, the analytical procedures for OTA determination in wines have to be both very sensitive and reliable. In this paper, we compared two quantification methods: the stable isotope dilution assay (SIDA) and the diastereomeric dilution assay (DIDA). For this purpose, non-natural analogues of OTA were synthesized: the labeled OTA (OTA-d4) as a diastereomeric mixture for the SIDA and one non-natural OTA's diastereomer (OTA-dia) for the DIDA. To quantify OTA in red grapes, musts or wines, the sample preparation was optimized using immunoaffinity column extraction and the analysis was performed by LC-MS/MS in Multiple Reaction Monitoring mode. A validation procedure in agreement with the International Organization of Vine and Wine recommendations was conducted. It appeared that SIDA quantification exhibited excellent sensitivity (LOD<1 ng L(-1)), accuracy (recovery=98%), repeatability (RSD<3%) and intermediate reproducibility (RSD<4%) compared to quantification by DIDA. Indeed, DIDA method did not provide satisfactory results demonstrating that immunoaffinity extraction is exclusively selective for the natural OTA and not for its diastereomer, which therefore cannot be considered as a good internal standard for this particular method.

First synthesis of a stable isotope of Ochratoxin A metabolite for a reliable detoxification monitoring.

Due to its toxicity and presence in numerous food products, Ochratoxin A (OTA) has drawn attention for decades. This article summarizes the first synthesis of a labeled analogue of Ochratoxin α (OTα), one of the main products generated by the metabolization of OTA by microorganisms. This synthesis also led to a new labeled analogue of OTA with the deuteration located on the dihydroisocoumarin moiety allowing thus both the accurate quantification of OTA and OTα and the establishing of a reliable detoxification rate.

Yeast-yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts.

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions.