New Insights Into Cinnamoyl Esterase Activity of Oenococcus oeni.

Some strains of Oenococcus oeni possess cinnamoyl esterase activity that can be relevant in the malolactic stage of wine production liberating hydroxycinnamic acids that are precursors of volatile phenols responsible for sensory faults. The objective of this study was to better understand the basis of the differential activity between strains. After initial screening, five commercial strains of O. oeni were selected, three were found to exhibit cinnamoyl esterase activity (CE+) and two not (CE-). Although the use of functional annotation of genes revealed genotypic variations between the strains, no specific genes common only to the three CE+ strains could explain the different activities. Pasteurized wine was used as a natural source of tartrate esters in growth and metabolism experiments conducted in MRS medium, whilst commercial trans-caftaric acid was used as substrate for enzyme assays. Detoxification did not seem to be the main biological mechanism involved in the activity since unlike its phenolic cleavage products and their immediate metabolites (trans-caffeic acid and 4-ethylcatechol), trans-caftaric acid was not toxic toward O. oeni. In the case of the two CE+ strains OenosTM and CiNeTM, wine-exposed samples showed a more rapid degradation of trans-caftaric acid than the unexposed ones. The CE activity was present in all cell-free extracts of both wine-exposed and unexposed strains, except in the cell-free extracts of the CE- strain CH11TM. This activity may be constitutive rather than induced by exposure to tartrate esters. Trans-caftaric acid was totally cleaved to trans-caffeic acid by cell-free extracts of the three CE+ strains, whilst cell-free extracts of the CE- strain CH16TM showed significantly lower activity, although higher for the strains in experiments with no prior wine exposure. The EstB28 esterase gene, found in the genomes of the 5 strains, did not reveal any difference on the upstream regulation and transport functionality between the strains. This study highlights the complexity of the basis of this activity in wine related O. oeni population. Variable cinnamoyl esterases or/and membrane transport activities in the O. oeni strains analyzed and a possible implication of wine molecules could explain this phenomenon.

Cheers, proost, saúde: Cultural, contextual and psychological factors of wine and beer consumption in Portugal and in the Netherlands.

Wine and beer consumption are an integral part of European culture: Southern Europe is associated with wine and Northern Europe is associated with beer. When consumed in moderation, these alcoholic beverages can be part of a balanced and healthy diet. In the 1990s, non-alcoholic beer (NAB), which has no cultural roots, became available in the market. This review identifies determinants for consumption of wine, beer, and NAB, using data on consumption patterns from Portugal and the Netherlands. Since the 1960s the image of Portugal as a wine country declined, whereas the image of the Netherlands as a beer country remained stable. In each country beer is now the most consumed alcoholic beverage and is mainly a men's beverage, whereas wine is the second most consumed and is consumed by both genders. Cultural differences define Portuguese as "outdoors, everyday drinkers", within a meal context, and Dutch as "at home, weekend drinkers." Wine is perceived as the healthiest beverage, followed by NAB, and regular beer. Motivation for consumption is related to context: wine for special occasions, beer for informal occasions, and NAB for occasions when alcohol is not convenient. Moderate wine and beer consumption seems to be surrounded by positive emotions. This review is relevant for public health, for industry market strategies, and identifies opportunities of future research on drinking behaviour.

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.

Production of volatile phenols by Lactobacillus plantarum in wine conditions.

Some lactic acid bacteria produce volatile phenols in culture medium but this activity has not been extensively studied in wine conditions. Red and white wines were mixed with MRS medium at different ratios to study the influence of wine on the metabolism of p-coumaric and ferulic acids by Lactobacillus plantarum. In MRS broth supplemented with these precursors at 10 mg l(-1), only 4-ethylphenol was produced (1 mg l(-1)) while, in the presence of wine, 4-vinylphenol was also obtained. Both volatile phenols are produced in nearly equal amounts (1 mg l(-1)) or almost only 4-vinylphenol depending on the MRS:wine ratio. Thus, wine favours the accumulation of 4-vinylphenol. Ferulic acid was not or was weakly metabolized in the conditions studied.

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.

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.

Determination of antioxidant capacity using the biological system bacteriophage P22/bacterium Salmonella typhimurium.

Bacteriophage/bacterium systems have been employed in the past in assays for virucidal activity. A novel application of one such system is proposed here for the in vivo determination of antioxidant capacity. It was shown that an antioxidant such as gallic acid can effectively protect against oxidative damage brought about by H2O2-but only within a narrow range of concentrations (i.e., from 250 to 500 mg L-1); ascorbic acid, on the other hand, did not exhibit any protective effect against H2O2. Finally, neither ascorbic nor gallic acid demonstrated a virucidal effect. The P22/Salmonella typhimurium model system thus proved to be useful in the assessment of antioxidant capacity in vivo, at least using those two alternative model antioxidants.

Phenotypic and genetic heterogeneity of lactic acid bacteria isolated from "Alheira", a traditional fermented sausage produced in Portugal.

The aim of this study was to evaluate the phenotypic and genetic heterogeneity of lactic acid bacteria (LAB) isolated from "Alheira", a fermented sausage produced in Portugal. LAB were identified to genus and species level by phenotypic characteristics, using genus or species-specific primers and sequencing of the gene encoding 16S rRNA. Two-hundred and eighty-three isolates were grouped into 14 species. Lactobacillus plantarum was isolated from all sausages and Enterococcusfaecalis from most of the samples. Low numbers of Lactobacillus paraplantarum, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus zeae, Lactobacillus paracasei, Leuconostoc mesenteroides, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella cibaria, Weissella viridescens and Enterococcus faecium were recorded. The genetic heterogeneity of L. plantarum and E. faecalis strains were determined by numerical analysis of DNA banding patterns obtained by RAPD-PCR. Strains of L. plantarum and E. faecalis were different from different producers. This study forms the basis from which starter cultures could be selected for production of "Alheira".

Impact of forced-aging process on madeira wine flavor.

The aim of this study was to determine the optimal temperature and baking time to obtain a Madeira wine considered typical by an expert panel. For this purpose simultaneous descriptive analyses of typical Madeira wines were performed, and seven descriptors were selected: "dried fruit", "nutty", "musty", "baked", "oak", "mushroom", and "brown sugar". Up to 10 odor-active zones were the most frequently cited by the members of the GC-olfactometry panel as corresponding to the panel's descriptors. The odor importance of each of the zones reported by the GC-O analysis was ranked by AEDA. Three odor zones were identified as common to both Malvasia and Sercial wines and had retention indices (RI) of 1993 ("brown sugar" and "toasted"), 2151 ("brown sugar"), and 2174 ("nutty", "dried fruits"); sotolon was identified as responsible for this last aroma. Several molecules were selected to be quantified on baked wines on the basis of AEDA results and expected Maillard volatiles, such as sotolon, furfural, 5-methylfurfural, 5-ethoximethylfurfural, methional, and phenylacetaldehyde. It was observed that typicity scores were positively correlated with the concentrations of sotolon and sugar and baking time and negatively with the fermentation length.

Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii.

The aim of this work was to investigate the effect of wine phenolic aldehydes, flavonoids and tannins on growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Cultures were grown in ethanol-containing MRS/TJ medium supplemented with different concentrations of phenolic aldehydes or flavonoids and monitored spectrophotometrically. The effect of tannins was evaluated by monitoring the progressive inactivation of cells in ethanol-containing phosphate buffer supplemented with grape seed extracts with different molecular weight tannins. Of the phenolic aldehydes tested, sinapaldehyde, coniferaldehyde, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde significantly inhibited the growth of O. oeni VF, while vanillin and syringaldehyde had no effect at the concentrations tested. Lact. hilgardii 5 was only inhibited by sinapaldehyde and coniferaldehyde. Among the flavonoids, quercetin and kaempferol exerted an inhibitory effect especially on O. oeni VF. Myricetin and the flavan-3-ols studied (catechin and epicatechin) did not affect considerably the growth of both strains. Condensed tannins (particularly tetramers and pentamers) were found to strongly affect cell viability, especially in the case of O. oeni VF. In general, this strain was found to be more sensitive than Lact. hilgardii 5 to the phenolic compounds studied. This work contributes to the knowledge of the effect of different phenolic compounds on the activity of wine lactic acid bacteria, which, especially in the case of aldehydes and of different molecular weight fractions of tannins, is very scarce.

Microbiological characterization of randomly selected Portuguese raw milk cheeses with reference to food safety.

Seventy raw milk cheeses made in different regions of Portugal, both hard and soft varieties, made with cow's, ewe's, or goat's milk or combinations of these, were sampled within their quoted shelf lives for microbiological safety. On the basis of the presence or numbers of Escherichia coli, E. coli O157, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, cheeses were categorized as satisfactory, acceptable, unsatisfactory, or unacceptable and potentially hazardous. Twenty-two of the 70 cheeses were classified as satisfactory or acceptable. Thirty-seven of the cheeses were considered unsatisfactory because of the presence of E. coli, S. aureus, or both, while 11 of the cheeses were graded as unacceptable and potentially hazardous because of the presence of excessive numbers of S. aureus, E. coli, or L. monocytogenes and the presence of Salmonella in three of these. All cheeses graded as unacceptable and potentially hazardous were soft or semisoft cheeses made with ewe's and goat's milk, with the exception of two hard cheeses made with cow's milk. E. coli O157 was not detected in any of the cheeses. According to the present results, it seems that the presence or counts of pathogenic or indicator organisms in raw milk cheeses cannot be related to the processing conditions, milk type, or region of production.

Chemical and microbiological characterisation of "Salpicão de Vinhais" and "Chouriça de Vinhais": traditional dry sausages produced in the North of Portugal.

"Chouriça de Vinhais" and "Salpicão de Vinhais" are traditional smoked naturally fermented meat products produced in the North of Portugal, Trás-os-Montes. The objective of this study was the characterisation of these products, giving particular attention to their microbiological and chemical safety. Nitrite, nitrate, heavy metals and biogenic amines were within accepted limits for meat products. Globally, the need for improvements in the good manufacturing practices was demonstrated in this study as various lots were considered of unsatisfactory microbiological quality according to the guidelines published by the Food Safety Authority of Ireland.

Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from "Alheira", a fermented sausage traditionally produced in Portugal.

Lactic acid bacteria were isolated from "Alheira" sausages that have been sampled from different regions in Portugal. The sausages were produced according to different recipes and with traditional starter cultures. Two isolates (HA-6111-2 and HA-5692-3) from different sausages were identified as strains of Pediococcus acidilactici. Each strain produces a bacteriocin, designated as bacHA-6111-2 and bacHA-5692-3. Both bacteriocins are produced at low levels after 18 h of growth in MRS broth (3200 AU/ml against Enterococcus faecium HKLHS and 1600 AU/ml against Listeria innocua N27). BacHA-6111-2 and bacHA-5692-3 are between 3.5 kDa and 6.5 kDa in size, as determined by tricine-SDS-PAGE. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of cell-free supernatants with proteinase K, pronase and trypsin. No change in activity was recorded when treated with catalase. Both bacteriocins are sensitive to treatment with Triton X-114 and Triton X-100, but resistant to Tween 20, Tween 80, SDS, Oxbile, NaCl, urea and EDTA. The bacteriocins remained stable after 2 h at pH 6.0. A decrease in antibacterial activity was recorded after 60 min at 100 degrees C. After 60 min at 80 degrees C, 60 degrees C and 25 degrees C the antibacterial activity against L. innocua N27 decreased by 25%. Addition of bacHA-6111-2 and bacHA-5692-3 (1600 AU/ml) to a mid-log (5-h-old) culture of L. innocua N27 inhibited growth for 7 h. Addition of the bacteriocins (3200 AU/ml) to a mid-log (5-h-old) culture of E. faecium HKLHS repressed cell growth. The bacteriocins did not adhere to the surface of the producer cells. Both strains contain a 1044 bp DNA fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragments from both bacteriocins revealed homology to large sections of pedA (188 bp), pedB (338 bp) and pedC (524 bp) of pediocin PA-1 and the bacteriocins are considered similar to pediocin PA-1.

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays.

A total of 226 lactic acid bacteria (LAB) isolated from "Alheira", a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from "Alheiras" and to select strains that could be used in "Alheira" production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of "Alheira mass" (sterilized), at 4°C. The growth of L. innocua population was significantly suppressed in the paste of "Alheira" when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1×10(7)CFU/g after 28 days of incubation).

Listeriosis in Portugal: an existing but under reported infection.

BACKGROUND: Listeriosis is a rare disease caused by the bacterium Listeria monocytogenes, the normal vehicle of which is food. The disease, which is largely confined to its risk groups of pregnant women, the elderly and immunocompromised individuals, has increased in incidence in recent years. In Portugal, listeriosis is not a notifiable infection and available data are scarce. The objective of this work was to collate the available information concerning listeriosis in Portugal by compiling a retrospective study of cases recorded over a decade. METHODS: Requests for case data on clinically confirmed listeriosis, recorded over the previous decade, were replied to by 23 hospitals and a National Institute of Health delegation. RESULTS: 35 cases of listeriosis were identified for the period between 1994 and 2003 inclusive, the mortality rate being greater than 17%. According to the data collected in this study for the year 2003, the incidence of this disease in Portugal was at least 1.4 cases per million inhabitants in that year. CONCLUSION: The study demonstrates, for the first time in the widely available literature, that despite their being no cases of listeriosis in Portugal recorded in official reports, the threat of L. monocytogenes to public health is of a similar dimension to that in other countries.

Chemical and microbiological characterization of alheira: A typical Portuguese fermented sausage with particular reference to factors relating to food safety.

Alheiras are traditional smoked naturally fermented meat sausages produced in the north of Portugal. They have not previously been characterized as to their chemical and microbiological status. pH and salt levels are insufficient to assure microbiological safety, there is ample opportunity for post-cooking contamination; the products require chill storage and cooking before consumption. Heavy metals and biogenic amines were, in general, within accepted limits for meat products. Lactic acid bacteria comprised the major microflora (ca. 7-8logcfu/g) with substantial counts of micrococci and enterococci (up to 7logcfu/g). Escherichia coli, Staphylococcus aureus and Listeria spp. were detected in several samples.

Alcohols, esters and heavy sulphur compounds production by pure and mixed cultures of apiculate wine yeasts.

Strains of Hanseniaspora uvarum, Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used as pure or mixed starter cultures in commercial medium, in order to compare their kinetic parameters and fermentation patterns. In pure and mixed cultures, yeasts presented similar ethanol yield and productivity. Pure cultures of H. uvarum and S. cerevisiae showed a specific growth rate of 0.38 h(-1); however, this value decreased when these yeasts were grown in mixed cultures with H. guilliermondii. The specific growth rate of pure cultures of H. guilliermondii was 0.41 h(-1) and was not affected by growth of other yeasts. H. guilliermondii was found to be the best producer of 2-phenylethyl acetate and 2-phenylethanol in both pure and mixed cultures. In pure cultures, H. uvarum led to the highest contents of heavy sulphur compounds, but H. guilliermondii and S. cerevisiae produced similar levels of methionol and 2-methyltetrahydrothiophen-3-one. Growth of apiculate yeasts in mixed cultures with S. cerevisiae led to amounts of 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2-methyltetrahydrothiophen-3-one similar to those obtained in a pure culture of S. cerevisiae; however, growth of apiculate yeasts increased methionol contents of fermented media.

Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces.

The heat resistance of three strains of Dekkera/Brettanomyces (Dekkera anomala PYCC 5,153, Dekkera bruxellensis PYCC 4,801 and Dekkera/Brettanomyces 093) was evaluated at different temperatures between 32.5 and 55 degrees C. Thermal inactivation tests were performed in tartrate buffer solution (pH 4.0) and in wines. In the studies employing buffer as the heating menstruum, measurable thermal inactivation began only at temperatures of 50 degrees C. When heating was performed in wine, significant inactivation begins at 35 degrees C. Subsequent thermal inactivation tests were performed in buffer at various levels of pH, ethanol concentration, and various phenolic acids. Results from experiments in buffer with added ethanol suggest that the greater heat sensitivity shown in wines can be largely attributed to ethanol, although potentiation of this effect might be due to the phenolic content, particularly from ferulic acid. In the range of pH values tested (2.5-4.5), this factor had no influence in the heat inactivation kinetics. Relevant data, in the form of D and Z values calculated in the various environments, potentially useful for the establishment of regimes of thermal control of Dekkera/Brettanomyces yeasts in wine and contaminated equipment is presented.

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation.

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.

Microbiological Characterization of Picante da Beira Baixa Cheese.

Ninety cheeses of the type Picante da Beira Baixa (a hard, highly salted, and spicy traditional cheese produced in Portugal from a mixture of goat's and ewe's milks) were manufactured and analyzed for microbiological quality at different stages of the 180-day ripening period. The most abundant species of Enterobacteriaceae , staphylococci, enterococci, lactobacilli, and yeasts found in the Picante cheese were Hafnia alvei and Serratia liquefaciens ; Staphylococcus hominis and S.xylosus ; Enteroccus faecium , E. faecalis , and E. durans ; Lactobacillus plantarum and L. paracasei ; and Debaryomyces hansenii and Yarrowia lipolytica .