Metabolism of Schizosaccharomyces pombe under reduced osmotic stress conditions afforded by fed-batch alcoholic fermentation of white grape must.

Strains of Schizosaccharomyces pombe are being increasingly investigated with regards to their grape winemaking potential either in combination with the typical production yeast, Saccharomyces cerevisiae, or in monoseptic fermentations. Their ethanol tolerance and ability to degrade L-malic acid is oenologically convenient but contrasts with the comparatively high acetic acid and acetaldehyde formation potential which is considered undesirable, especially in white winemaking. The purpose of this work was to investigate the performance of a selected S. pombe strain in monoseptic femerntations of white grape must. Traditional batch fermentations were compared with an innovative and automated fed-batch fermentation technique were sugar concentrations are kept low during fermentations to decrease sugar induced osmotic stress. Because of its known effect on growth and ethanol tolerance, the effect of Mg was also tested. While Mg supplementation was not shown to significantly influence residual values of sugars, ethanol, glycerol, organic acids and acetaldehyde, the application of the fed-batch technique led to a fundamental change in yeast physiology. While glycerol values were only slightly reduced, the fed-batch approach allowed obtaining wines devoid of acetic acid whose levels were considerable in wines produced by the traditional batch technique (0.6 g/L). The work demonstrates that the acetic acid metabolism of S. pombe is associated to sugar induced osmotic stress such as for S. cerevisiae, too, and may be controlled by application of suitable fermentation techniques for winemaking.

Improved sample preparation and rapid UHPLC analysis of SO2 binding carbonyls in wine by derivatisation to 2,4-dinitrophenylhydrazine.

Sulphur dioxide (SO2) is essential for the preservation of wines. The presence of SO2 binding compounds in musts and wines may limit sulphite efficacy leading to higher total SO2 additions, which may exceed SO2 limits permitted by law and pose health risks for sensitive individuals. An improved method for the quantification of significant wine SO2 binding compounds is presented that applies a novel sample treatment approach and rapid UHPLC separation. Glucose, galacturonic acid, alpha-ketoglutarate, pyruvate, acetoin and acetaldehyde were derivatised with 2,4-dinitrophenylhydrazine and separated using a solid core C18 phase by ultra high performance liquid chromatography. Addition of EDTA to samples prevented de novo acetaldehyde formation from ethanol oxidation. Optimised derivatisation duration enhanced reproducibility and allowed for glucose and galacturonic acid quantification. High glucose residues were found to interfere with the recovery of other SO2 binders, but practical SO2 concentrations and red wine pigments did not affect derivatisation efficiency. The calibration range, method accuracy, precision and limits of detection were found to be satisfactory for routine analysis of SO2 binders in wines. The current method represents a significant improvement in the comprehensive analysis of SO2 binding wine carbonyls. It allows for the quantification of major SO2 binders at practical analyte concentrations, and uses a simple sample treatment method that prevents treatment artifacts. Equipment utilisation could be reduced by rapid LC separation while maintaining analytical performance parameters. The improved method will be a valuable addition for the analysis of total SO2 binder pools in oenological samples.

Metabolism of SO2 binding compounds by Oenococcus oeni during and after malolactic fermentation in white wine.

Sulfur dioxide SO2 is the key additive for the preservation of wines. Carbonyl and keto compounds in wine can bind to SO2 and decrease its efficacy, resulting in higher total SO2 requirements. Increased consumer demand for low sulfite and organic wines pose production challenges if SO2 binders have not been properly managed during vinification. Malolactic fermentation (MLF) has been known to reduce bound SO2 levels but detailed time course studies are not available. In this work, the kinetics of major SO2 binding compounds and malic acid were followed during MLF in wine with 12 commercially available strains of Oenococcus oeni. Pyruvic acid, acetaldehyde and α-ketoglutaric acid were degraded to various degrees by O. oeni, but galacturonic acid was not. At the time of malic acid depletion, percent degradation of pyruvate, α-ketoglutaric acid and acetaldehyde was 49%, 14% and 30%, respectively. During MLF, the decrease in average bound SO2 levels, as calculated from carbonyl metabolism, was 22%. The largest reduction in wine carbonyl content occurred in the week after completion of MLF and was 53% (107 mg/L to 34 mg/L) calculated as bound SO2. Prolonged activity of bacteria in the wines (up to 3 weeks post malic acid depletion) resulted only in reduced additional reductions in bound SO2 levels. The results suggest that microbiological wine stabilization one week after malic acid depletion is an effective strategy for maximum removal of SO2 binders while reducing the risk of possible post-ML spoilage by O. oeni leading to the production acetic acid and biogenic amines.

A rapid method for the determination of microbial biomass by dry weight using a moisture analyser with an infrared heating source and an analytical balance.

AIMS: Microbial biomass is an important biotechnological parameter. The traditional method for its determination involves an oven-drying step and equilibration to room temperature before weighing, and it is tedious and time consuming. This work studied the utilisation of a moisture analyser consisting of an efficient infrared-heating module and an analytical balance for the determination of microbial biomass by dry weight. METHODS AND RESULTS: The method duration depended on the sample volume and was between 7 and 40 min for sample volumes of 1-10 ml. The method precision depended on the total dry weight analysed - 10 mg of total dry weight being sufficient to achieve coefficients of variation of 5% or less. Comparison with the conventional oven method provided a correlation coefficient r(2) of 0.99. The recovery of an internal standard ranged between 94.2 and 106.4% with a precision of 1.39-4.53%CV. CONCLUSIONS: Validation revealed sufficient method accuracy, precision and robustness and was successfully applied to the study of yeast and bacterial growth kinetics. Techniques are discussed that allow for increased method precision at low biomass concentrations, and equations are provided to estimate required drying time and method precision based on sample volume and total sample dry weight, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents a rapid method for the determination of microbial biomass, allowing for the timely implementation of biomass-based information in biotechnological and laboratory protocols.

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation.

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. CONCLUSIONS: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.

Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine.

AIMS: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO(2). The current work investigated the degradation of acetaldehyde and SO(2)-bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. METHODS AND RESULTS: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to pH 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO(2)-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. CONCLUSIONS: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO(2)-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO(2) should be considered next to free SO(2) in order to evaluate malolactic fermentability. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides new results regarding the metabolism of acetaldehyde and SO(2)-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO(2).

Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations.

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.

Quantitative determination of L-arginine by enzymatic end-point analysis.

An enzymatic end-point method for the quantitative determination of L-arginine was evaluated with samples of synthetic wine and natural grape juice. The enzymes arginase, urease, and glutamate dehydrogenase were used in this simple assay, similar to those described for many metabolites by Boehringer-Mannheim. In synthetic wine, recovery of L-arginine ranged between 98.3 and 104.4% and the precision as coefficient of variation was between 0.4 and 1.47% in the concentration range of the method, 0-100 mg/L L-arginine. The recovery of L-arginine in a grape juice with added L-arginine after clarification with polyvinylpolypyrrolidone ranged between 100 and 101.3%, and the coefficient of variation was 0.6%. The method has low material costs of approximately 0.43 U.S.$ per assay, and the time course of the reaction facilitates measurement of several samples concurrently. The results of this evaluation indicate that the enzymatic assay is a preferred method over colorimetric methods for the manual determination of L-arginine.

Kinetics of the arginine metabolism of malolactic wine lactic acid bacteria Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo111.

The excretion of citrulline, a precursor of carcinogenic ethyl carbamate, formed from arginine degradation by malolactic bacteria in wine is of toxicological concern. The arginine metabolism of resting cells of Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo1l1 was examined. The citrulline excretion rate was found to be linearly correlated to the arginine degradation rate. It was possible to calculate an arginine to citrulline conversion ratio which could be used to predict the amount of citrulline expected after the degradation of a known quantity of arginine. The conversion ratios determined in this study were similar to data calculated from other authors for fermentations in wine and ranged between 4.0% and 7.7%. Ribose, fructose and glucose inhibited the degradation of arginine in Lact. buchneri CUC-3, and inhibition of arginine degradation by glucose correlated with higher arginine to citrulline conversion ratios. The work presents new results of arginine metabolism in malolactic bacteria and gives starting points for investigations in wine.

Acetaldehyde metabolism by wine lactic acid bacteria.

Acetaldehyde is a volatile flavor compound present in many fermented foods and is important in the production of red and white wines. Nine strains of the genera Lactobacillus and Oenococcus were able to metabolize acetaldehyde in a resting cell system, whereas two Pediococcus strains were not. Acetic acid and ethanol were produced from its degradation. A Lactobacillus and an Oenococcus were able to degrade SO(2)-bound acetaldehyde, as well. A coincubation of resting cells of Saccharomyces bayanus Première Cuvée and Oenococcus oeni Lo111 showed that strain Lo111 metabolized acetaldehyde produced by the yeast. The ability of malolactic bacteria to degrade free and SO(2)-bound acetaldehyde has implications for sensory and color qualities and the use of SO(2) in wine.

Intracellular glucose 6-phosphate content in Streptomyces coelicolor upon environmental changes in a defined medium.

A new, chemically defined medium providing dispersed growth and high biomass formation and a method for quantitative extraction of intracellular metabolites was used to investigate the cellular response of Streptomyces coelicolor A3(2) during growth and upon changes in nutrient utilization. Fast changes of the glucose 6-phosphate content precisely signaled transitions in the flow of carbon sources. The results indicate that intracellular pool sizes may be used to detect early nutrient limitations in view of the onset of antibiotic production. Additionally the results disclose characteristics of the regulation of maltose and glutamic acid uptake and degradation in S. coelicolor A3(2).

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria.

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.