Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria.

AIMS: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4-vinylphenol [4VP] and 4-ethylphenol [4EP]) from the metabolism of p-coumaric acid by lactic acid bacteria (LAB). METHODS AND RESULTS: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p-coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p-coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l(-1) ) of tannins inhibited the synthesis of 4VP by Lact. plantarum. CONCLUSIONS: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p-coumaric acid. On the other hand, tannins exert an inhibitory effect. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.

Cell membrane damage induced by phenolic acids on wine lactic acid bacteria.

The aim of this work was to investigate the effect of phenolic acids on cell membrane permeability of lactic acid bacteria from wine. Several phenolic acids were tested for their effects on the cell membrane of Oenococcus oeni and Lactobacillus hilgardii by measuring potassium and phosphate efflux, proton influx and by assessing culture viability employing a fluorescence technique based on membrane integrity. The experimental results indicate that hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids) induce greater ion leakages and higher proton influx than hydroxybenzoic acids (p-hydroxibenzoic, protocatechuic, gallic, vanillic, and syringic acids). Among the hydroxycinnamic acids, p-coumaric acid showed the strongest effect. Moreover, the exposure of cells to phenolic acids caused a significant decrease in cell culture viability, as measured by the fluorescence assay, in both tested strains. The results agree with previous results obtained in growth experiments with the same strains. Generally, phenolic acids increased the cell membrane permeability in lactic acid bacteria from wine. The different effects of phenolic acids on membrane permeability could be related to differences in their structure and lipophilic character.

Physiological responses to atmospheric fluorine pollution in transplants of Pseudoscleropodium purum.

Transplants of the moss Pseudoscleropodium purum were placed under shade, and with an auto irrigation system, at five sites in the surroundings of an aluminium smelter to enable study of the possible effects of F on different physiological parameters after exposure periods of 28 and 56 days. Reference thresholds for the content of Chl a+b and for the pigment indexes of Chl a/b and D665/D665a were established with the data corresponding to 22 transplants of each duration of exposure, at seven sites where there was little or no contamination by F. The highest determination coefficients for the levels of F in moss were those obtained for chlorophyll content (Chl a+b) and the D665/D665a pigment index, with the effects being most evident in the transplants exposed to the contamination for 56 days.

Testing differences in methods of preparing moss samples.

Various methods are currently used to prepare moss samples prior to their analysis in biomonitoring surveys. To date there has been no investigation of whether different results are obtained by using different preparation techniques. We therefore selected three preparation procedures (freezing, drying and acclimatization), currently applied in the European Moss Survey and other studies, to test whether significantly different concentrations of Al, As, Cd, Cr, Cu, Fe, Hg, K, Ni, Pb, Se, V and Zn are obtained. In addition, we considered the effect that the degree and type of contamination of the sampling sites and the different times of the year may have on the results. We found significant differences in the concentrations of all elements in the moss samples subjected to the different procedures and recommend standardization of the methods of sample preparation for valid comparison of the results. We further recommend that the samples are picked over directly in the field, to remove adhering material, and then cleaned prior to analysis.

Definition and number of subsamples for using mosses as biomonitors of airborne trace elements.

We collected 50 subsamples of the moss Scleropodium purum from each of three sampling sites and determined the concentrations of As, Cd, Cu, K, Hg, Ni, Pb, Se, and Zn in each subsample. We then calculated the number of subsamples required to determine significant differences in the mean concentrations of two sampling sites. We found that to differentiate between an uncontaminated sampling site and another, slightly contaminated site, 30 subsamples are required from each. On the basis of these results and because, to date, there are no studies that justify the application of the previously proposed recommendations, studies of local variability in other areas, and under different conditions, with other contaminants and moss species must be undertaken. For posterior comparison of data on variability as well as application of the recommendations associated with such results, the general use of a single definition of a subsample is required. We propose the following definition of a subsample: "1 g dry weight (approx.), collected within a 25-cm radius of a node, selected at random from a 1 x 1-m sampling grid placed in a sampling site."

A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines.

AIMS: The development of a simple and reliable procedure, compatible with routine use in wineries, for the presumptive detection of Brettanomyces/Dekkera from wine and wine-environment samples. METHODS AND RESULTS: The method of detection of these yeasts employs a selective enrichment medium. The medium contains glucose (10 g l(-1)) as carbon and energy source, cycloheximide (20 mg l(-1)) to prevent growth of Saccharomyces, chloramphenicol (200 mg l(-1)) to prevent growth of bacteria and p-coumaric acid (20 mg l(-1)) as the precursor for the production of 4-ethyl-phenol. After the inoculation with wine, the medium is monitored by visual inspection of turbidity and by periodic olfactive analysis. Contaminated wines will develop visible turbidity in the medium and will produce the 4-ethyl-phenol off-odour, which can be easily detected by smelling. CONCLUSIONS: A selective enrichment liquid medium was developed to differentially promote the growth and activity of Brettanomyces/Dekkera. The method is simple to execute, employing a simple-to-prepare medium and a periodic olfactive detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of the procedure make it particularly applicable in a wine-making environment thus presenting important advantages to the wine industry.

The effect of sampling design on extensive bryomonitoring surveys of air pollution.

In this study, we investigated if the different sampling designs normally used in extensive bryomonitoring surveys of air pollution allow compliance with the objectives proposed in the 'Heavy Metals in European Moss' project. For this, we have used data on concentrations of As, Cu, Se and Zn from five moss sampling surveys carried out in Galicia (NW Spain), using three types of sampling design: regular grid, sampling sites inside squares and the strategy recommended in the guidelines for European surveys. To study the effect of the interaction between the sampling design and the spatial pattern of the presence of contaminants, we have used a simulated and a real pattern onto which we superimposed the three sampling designs considered. We found that only random sampling and grid sampling allow compliance with the proposed objectives, and we discuss the advantages of the latter over the former. The data corresponding to studies using different sampling designs are not comparable, and in future large-scale surveys, the same design must be used if the results are to be compared and if valid conclusions are to be reached. The scale of the grid determines the scale to which the objectives are complied with, so that for correct standardization of the technique, and taking into account the most commonly used grid sizes, we recommend a grid size of 30 x 30 or 32 x 32 km. When additional samplings are required, the size of the grid size should be 30/2(n) or 32/2(n) km.

A new method for testing the sensitivity of active biomonitoring: an example of its application to a terrestrial moss.

For the first time, the concepts of limit of detection and limit of quantification, commonly used in analytical chemistry, are applied to the field of active biomonitoring with terrestrial mosses, using the controls as blanks so that the limits indicate the error associated with the transplant technique. The application of these concepts to data corresponding to Hg concentrations in the surroundings of a chlor-alkali plant and a power plant, makes interpretation of the results easier by providing better spatial and temporal coherence. This procedure may allow improvement in the standardization of active biomonitoring techniques as it is applicable to all kinds of biomonitors. One disadvantage of the application of the limit of detection and limit of quantification is that they only take into account the alpha error, or risk of false positives, and do not take into account the beta error, or risk of false negatives.

Active biomonitoring of element uptake with terrestrial mosses: a comparison of bulk and dry deposition.

Moss (Scleropodium purum) transplants were used to study bioconcentration originating from dry and bulk deposition, by measuring the tissue contents of Al, As, Ca, Cu, Fe, Hg, Mn, Ni, Pb, Se and Zn. Furthermore, a laboratory experiment was carried out to determine the sequence of maximum concentration and affinity of Al, Cu, Fe, Hg, Ni, Pb and Zn, in S. purum. We found that in many of the transplants, for the same period of exposure, higher levels of metals were accumulated via dry deposition than via bulk deposition. This result may be explained by the simple washing action of the rain on the surface of the moss, and by the existence of processes that provoke the loss of some of the accumulated elements: intercationic displacement and leaching caused by acid precipitation. Modelling of the final bioconcentration observed, as a balance of inputs and outputs of elements, revealed that this terrestrial moss does not integrate, but rather concentrates atmospheric deposition, and there exists a state of unstable equilibrium between inputs and outputs of elements, a state that is determined by the characteristics of the surrounding environment. On the basis of the results obtained in this study, we can conclude that at present, it is not possible to extrapolate the calibrations between the concentrations of elements accumulated in a certain species of moss to values of atmospheric deposition (bulk deposition) from one place to another with different environmental conditions.

Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii.

AIMS: To determine the effect of several wine-associated, phenolic acids on the growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. METHODS AND RESULTS: Growth was monitored in ethanol-containing medium supplemented with varying concentrations of hydroxybenzoic acids (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids). Progressive inactivation was monitored in ethanol-containing phosphate buffer supplemented in a similar manner to the growth experiments. Hydroxycinnamic acids proved to be more inhibitory to the growth of O. oeni than hydroxybenzoic acids. On the other hand, some acids showed a beneficial effect on growth of Lact. hilgardii. p-Coumaric acid showed the strongest inhibitory effect on growth and survival of both bacteria. CONCLUSIONS: Most phenolic acids had a negative effect on growth of O. oeni, for Lact. hilgardii this effect was only noted for p-coumaric acid. Generally, O. oeni was more sensitive to phenolic acid inactivation than Lact. hilgardii. SIGNIFICANCE AND IMPACT OF THE STUDY: Eight wine-derived, phenolic acids were compared for their effects on wine lactic acid bacteria. Results indicate that phenolic acids have the capacity to influence growth and survival parameters. The differences found between phenolic compounds could be related to their different chemical structures.

Citrulline as the main precursor of ethyl carbamate in model fortified wines inoculated with Lactobacillus hilgardii: a marker of the levels in a spoiled fortified wine.

AIMS: The aim of this study was to investigate the production of ethyl carbamate (EC) precursors by Lactobacillus hilgardii in model and Douro fortified wines and to determine the relationship between these compounds and EC levels in this type of wine. METHODS AND RESULTS: Several model fortified wines and fortified wine inoculated with L. hilgardii were analysed for citrulline and EC formation. A good correlation (R > 0.9) was obtained between citrulline and potential EC (that EC which is formed during heating of sample at 80 degrees C for 48 h). CONCLUSIONS: This correlation allowed us to calculate the potential EC formed during lactic acid bacteria activity in fortified wine. SIGNIFICANCE AND IMPACT OF THE STUDY: A good correlation was obtained (R=0.92) between measured and calculated EC in spoiled fortified wines, citrulline apparently being the main EC precursor produced by Lact. hilgardii thus contributing to the potential EC in this type of wine.