Francisco M. Campos

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.

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine.

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.

Factors influencing the production of volatile phenols by wine lactic acid bacteria

This work aimed to evaluate the effect of certain factors on the production of volatile phenols from the metabolism of p-coumaric acid by lactic acid bacteria (LAB) (Lactobacillus plantarum, L. collinoides and Pediococcus pentosaceus). The studied factors were: pH, L-malic acid concentration, glucose and fructose concentrations and aerobic/anaerobic conditions. It was found that, in the pH range of 3.5 to 4.5, the higher the pH the greater the production of volatile phenols. This behaviour is correlated with the effect of pH on bacterial growth. Increasing levels of L-malic acid in the medium diminished the production of 4-vinylphenol (4VP) and stimulated the production of 4-ethylphenol (4EP) by L. plantarum NCFB 1752 and L. collinoides ESB 99. The conversion of 4VP into 4EP by the activity of the vinylphenol reductase may be advantageous to the cells in the presence of L-malic acid, presumably due to the generation of NAD+, a cofactor required by the malolactic enzyme. Relatively high levels of glucose (20 g/L) led to an almost exclusive production of 4VP by L. plantarum NCFB 1752, while at low concentrations (≤ 5 g/L), 4EP is mainly or solely produced. Part of the glucose may be diverted to the production of mannitol as an alternative pathway to regenerate NAD+. This is corroborated by the experiments done with fructose, a compound that can be used as an electron acceptor by some bacteria becoming reduced to mannitol. In anaerobiosis, the reduction of 4VP into 4EP is clearly favoured, which is consistent with the need to increase the availability of NAD+ in these conditions. This study shows that the amount and the ratio 4VP/4EP produced by LAB are greatly affected by certain environmental and medium composition factors. The behaviour of the bacteria seems to be driven by the intracellular NAD+/NADH balance.

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).

Activity of lysozyme on Lactobacillus hilgardii strains isolated from Port wine.

This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme.

Utilisation of Natural and By-Products to Improve Wine Safety

Although wine is considered a relatively safe food product, there are a number of hazards associated with the use of certain additives and with some microbial metabolites produced during the winemaking process. Particularly, sulphur dioxide (SO2) may be responsible for adverse health effects in a small but significant proportion of the population. Thus, SO2 reduction or replacement is a major driver in the search for new antimicrobial and antioxidant agents. In this context, there is a growing interest in the antimicrobial properties of naturally existing products, which could be applied in the winemaking process. In this chapter we discuss the current knowledge about the effects of natural antimicrobial products (both intrinsic and extrinsic to wine composition) that can be used to minimise some health risks in the context of food safety. The role of phenolic compounds is emphasised since they seem to play a pivotal role in the antimicrobial activities of plant-derived materials but the use of proteins (lysozyme, bacteriocins and peptides) is also discussed. Finally the state of the art of the use of natural extracts in winemaking context is reviewed.