Correlation between IRC7 gene expression and 4-mercapto-4-methylpentan-2-one production in Saccharomyces cerevisiae strains.

Volatile thiols are not present in must but are synthesized and released by wine yeasts during alcoholic fermentation. In this study, autochthonous and commercial Saccharomyces cerevisiae strains were characterized for the expression of the main genes involved in thiols metabolism and their production in wine. New primer sets were developed on the basis of the S288c genome to evaluate the expression of Cys3, Cys4, MET17 and IRC7 genes. Obtained data revealed the occurrence of some thiols, for example, 4-mercapto-4-methylpentan-2-one (4-MMP) and 3-mercaptohexan-1-ol (3-MH) in Pecorino white wine. All genes were upregulated, but only for IRC7 was found a correlation with 4-MMP release: strains with the highest production showed the highest transcription level. IRC7 gene could be proposed as target for the selection of S. cerevisiae strains to increase thiols content in wine.

ß-Hydroxy ß-methylbutyrate free acid alters cortisol responses, but not myofibrillar proteolysis, during a 24-h fast.

This study was a randomised, double-blind, placebo-controlled cross-over trial examining the effects of ß-hydroxy ß-methylbutyrate free acid (HMB-FA) supplementation on muscle protein breakdown, cortisol, testosterone and resting energy expenditure (REE) during acute fasting. Conditions consisted of supplementation with 3 g/d HMB-FA or placebo during a 3-d meat-free diet followed by a 24-h fast. Urine was collected before and during the 24-h fast for analysis of 3-methylhistidine:creatinine ratio (3MH:CR). Salivary cortisol, testosterone, their ratio (T:C), and the cortisol awakening response were assessed. ANOVA was used to analyse all dependent variables, and linear mixed models were used to confirm the absence of carryover effects. Eleven participants (six females, five males) completed the study. Urinary HMB concentrations confirmed compliance with supplementation. 3MH:CR was unaffected by fasting and supplementation, but the cortisol awakening response differed between conditions. In both conditions, cortisol increased from awakening to 30 min post-awakening (P=0·01). Cortisol was reduced from 30 to 45 min post-awakening with HMB-FA (-32 %, d=-1·0, P=0·04), but not placebo (PL) (-6 %, d=-0·2, P=0·14). In males, T:C increased from 0 to 24 h of fasting with HMB-FA (+162 %, d=3·0, P=0·001), but not placebo (+13 %, d=0·4, P=0·60), due to reductions in cortisol. REE was higher at 24 h of fasting than 16 h of fasting independent of supplementation (+4·0 %, d=0·3, P=0·04). In conclusion, HMB-FA may affect cortisol responses, but not myofibrillar proteolysis, during acute 24-h fasting.

Dietary supplementation of branched-chain amino acids increases muscle net amino acid fluxes through elevating their substrate availability and intramuscular catabolism in young pigs.

Branched-chain amino acids (BCAA) have been clearly demonstrated to have anabolic effects on muscle protein synthesis. However, little is known about their roles in the regulation of net AA fluxes across skeletal muscle in vivo. This study was aimed to investigate the effect and related mechanisms of dietary supplementation of BCAA on muscle net amino acid (AA) fluxes using the hindlimb flux model. In all fourteen 4-week-old barrows were fed reduced-protein diets with or without supplemental BCAA for 28 d. Pigs were implanted with carotid arterial, femoral arterial and venous catheters, and fed once hourly with intraarterial infusion of p-amino hippurate. Arterial and venous plasma and muscle samples were obtained for the measurement of AA, branched-chain α-keto acids (BCKA) and 3-methylhistidine (3-MH). Metabolomes of venous plasma were determined by HPLC-quadrupole time-of-flight-MS. BCAA-supplemented group showed elevated muscle net fluxes of total essential AA, non-essential AA and AA. As for individual AA, muscle net fluxes of each BCAA and their metabolites (alanine, glutamate and glutamine), along with those of histidine, methionine and several functional non-essential AA (glycine, proline and serine), were increased by BCAA supplementation. The elevated muscle net AA fluxes were associated with the increase in arterial and intramuscular concentrations of BCAA and venous metabolites including BCKA and free fatty acids, and were also related to the decrease in the intramuscular concentration of 3-MH. Correlation analysis indicated that muscle net AA fluxes are highly and positively correlated with arterial BCAA concentrations and muscle net BCKA production. In conclusion, supplementing BCAA to reduced-protein diet increases the arterial concentrations and intramuscular catabolism of BCAA, both of which would contribute to an increase of muscle net AA fluxes in young pigs.

Presence of Disulfide-Bonded Thiols in Malt and Hops as the Precursors of Thiols in Beer.

Disulfide-bonded thiols in malt and hops were first identified as possible precursors of thiols in beer. The presence of disulfide-bonded 3-mercaptohexan-1-ol (3MH) was confirmed in malt and hops by observing an 8.9-9.9 times increase in the 3MH concentration in hopped water and unhopped wort after the reduction using tris(2-carboxyethyl)phosphine (TCEP), a reducing agent specific for disulfide bonds. The presence of disulfide-bonded 4-mercapto-4-methylpentan-2-one (4MMP) was confirmed in hops by observing 2.1 and 5.1 times increase in the 4MMP concentration after reduction in hopped water. Proteins, peptides, and amino acids having sulfhydryl groups or other thiol substances were assumed to form disulfide bonds with polyfunctional thiols in malt and hops. The release of thiols by the reduction of disulfide-bonded thiols during fermentation was first identified. A 65-82% of disulfide-bonded 3MH were reduced during fermentation, and as a result, concentrations of 3MH in hopped water and unhopped wort increased by 9.5-14.2 times during fermentation.

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.

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.