Characterization of phenolic acid reductase and decarboxylase activities of lactic acid bateria

Hydroxycinnamic acids are natural constituents of grape juice and wine, and are precursors of volatile phenols produced by yeasts and lactic acid bacteria (LAB). The organoleptic defects due to the presence of this volatile phenols are usually associated with “animal”, “horsey”, “leather”, “phenolic” or “spicy” aromatic notes. The most common pathway for the degradation of hydroxycinnamic acids involves two enzymes. In first place, it occurs a decarboxylation by the phenolic acid decarboxylase (PAD), and secondly a reduction of the intermediate metabolite carried out by the vinylphenol reductase (VPR). Some LAB strains seem to be able to metabolise hydroxycinnamic acids through an alternative route, involving a reduction step by a phenolic acid reductase (PAR) followed by a decarboxylation reaction. In order to characterize PAR and PAD/VPR activities, six different LAB strains were tested for the production of volatile phenols and non-volatile metabolites. The main objective of this work was to study the influence of certain factors/growth conditions on the extent of these activities. In the first part of this study, we evaluated the influence of the concentration of p-coumaric acid (1.0, 5.0 and 50 mg/L) as precursor for the production of volatile phenols. In the second part, the effect of the presence of L-malic acid and fructose on PAR and PAD/VPR activities with regard to the metabolism of p-coumaric acid was studied. The results show that all of the LAB strains tested have the ability to produce volatile phenols, however, strain-dependent patterns were observed. Four of the strains were also found to produce phloretic acid from 50 mg/l of p-coumaric acid using the alternative metabolic route mentioned above, thus indicating PAR activity. L-malic acid and fructose seemed to stimulate VPR activity, while no alteration was observed on the PAR activity. Moreover, the initial concentration of p-coumaric acid used seems to play an important role on volatile phenols production

Two decades of "Horse sweat" taint and Brettanomyces yeasts in wine: where do we stand now ?

ReviewThe unwanted modification of wine sensory attributes by yeasts of the species Brettanomyces bruxellensis due to the production of volatile phenols is presently the main microbiological threat to red wine quality. The effects of ethylphenols and other metabolites on wine flavor is now recognized worldwide and the object of lively debate. The focus of this review is to provide an update of the present knowledge and practice on the prevention of this problem in the wine industry. Brettanomyces bruxellensis, or its teleomorph, Dekkera bruxellensis, are rarely found in the natural environment and, although frequently isolated from fermenting substrates, their numbers are relatively low when compared with other fermenting species. Despite this rarity, they have long been studied for their unusual metabolical features (e.g., the Custers effect). Rising interest over the last decades is mostly due to volatile phenol production affecting high quality red wines worldwide. The challenges have been dealt with together by researchers and winemakers in an effective way and this has enabled a state where, presently, knowledge and prevention of the problem at the winery level is readily accessible. Today, the main issues have shifted from technological to sensory science concerning the effects of metabolites other than ethylphenols and the over estimation of the detrimental impact by ethylphenols on flavor. Hopefully, these questions will continue to be tackled together by science and industry for the benefit of wine enjoymentinfo:eu-repo/semantics/publishedVersio

Selective use of wine yeast strains having different volatile phenols production

Among Saccharomyces cerevisiae wine yeasts, we found a high frequency of strains having the ability to decarboxylate 4-hydroxycinnamic acid and 3-methoxy-4-hydroxy-cinnamic acid. From Gewurztraminer juices fermented by S. cerevisiae wine strains with and without such character, we obtained wines with considerably different levels of volatile phenols and some interesting evidences of the likely precursors of 4-vinylguaiacol and 4-vinylphenol. The identification of yeast strains by electrophoretic karyotyping gave us the possibility of evaluating the effective contribution of the yeast in the organoleptic characteristic of Traminer wines associated with the concentration of such volatile phenols

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols

Volatile phenols in aged wine spirits: role, contents and impact of ageing systems

The volatile phenols (eugenol, guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol and 4-allylsyringol) are odorant compounds that may exist in aged wine spirits resulting from their contact with wooden barrels during the ageing process. These compounds, which are originated from wood lignin’s, revealed an important sensory impact in aged wine spirits due to their low sensory thresholds and correlations with sensory attributes such as woody, toasted, smoke, which have a close relationship with the quality of these beverages. The wine spirits are traditionally aged in wooden barrels but the use of wood fragments, with or without micro-oxygenation, is a technological alternative that has been recently studied by our team with promising results. This work presents an overview of volatile phenols’ amounts in wine spirits aged in wooden barrels during different ageing times and using two kinds of wood (chestnut versus oak). These compounds were quantified by GC-FID, after a previous extraction and concentration steps, and their identification was assessed by GC-MS. It is also examined the results and the impact of alternative technologies on the amounts of such compounds. The ANOVA results showed a significant effect of the ageing system and the wood botanical species on the volatile phenols contentsinfo:eu-repo/semantics/publishedVersio

Extraction of wood compounds by use of subcritical fluids

A study of the extraction of oak wood compounds with subcritical water-ethanol mixtures as extractant, with an ethanol content between 0-60%, is reported. Identification and characterisation of the extracted compounds have been made by spectrophotometry and gas chromatography with either flame ionisation or mass detectors. Extraction was performed statically manner by use of a single cycle or repeated cycles. All variables affecting the extraction process were studied and optimised. Extraction time and temperature are 60 min and 200ºC, respectively. Comparison of the extract thus obtained with commercial extracts showed the former to be rich in compounds characteristic of the commercial extracts. The method enables manipulation of the extract composition by changing the temperature and water/ethanol ratio used. It is faster than the traditional procedures for obtaining wood extracts

Use of superheated liquids for the extraction of non-volatile compounds from wood: HPLC studies

A study of the extraction of oak wood compounds using superheated water-ethanol mixtures ranging from 10 to 60% ethanol is reported. Identification and characterization of the extracted compounds have been made by high performance liquid chromatography. The extraction has been performed using the static mode by single or repetitive cycles. The variables affecting the extraction process have been studied and their optimum values established (extraction time: 50 min; pressure: 40 atm; extraction temperature: 180º C). The study allows to compare the non-volatile polyphenol fractions obtained in this way with those present in commercial samples with fully agreement between them. In addition, the method allows manipulation of the extract composition by changing the working pressure, temperature and water-ethanol ratio

The influence of grape variety on the production of volatile phenols in Portuguese wines

The main purpose of this work is to determine whether there is a correlation between the susceptibility to volatile phenol production of wines and grape variety. Therefore, 11 single varietal red wines from Portugal were heat sterilized, contaminated with Dekkera bruxellensis PYCC 4801 and incubated for at least 10 days at 30°C. Since yeasts did not grow in pure wines due to inhibition by ethanol, the experiments were conducted in diluted samples with similar initial pH and ethanol levels. Yeast growth was monitored and produced levels of 4-ethylguaiacol, 4-ethylphenol and 4-vinylphenol were measured using gas chromatography in order to compare values between varieties. This approach led to the production of 4-ethylphenol and 4-ethylguaiacol in all samples; 4-vinylphenol however, could not be detected in any sample. All values reached or exceeded perception threshold levels established in literature. This suggests that all examined wines are at risk of developing phenolic off-flavour once contaminated with Brettanomyces/Dekkera yeasts, especially considering that samples were diluted and thus contained lower amounts of precursors than are potentially present in pure wines. 4-ethylguaiacol values were generally lower and varied less between samples and varieties compared to 4-ethylphenol. Tinta Roriz and Touriga Franca showed the highest potential in regard to volatile phenol production from the natural precursors available in the wines while Sousão wines appeared to be the least prone and Touriga Nacional wines exhibited intermediate volatile phenol values. Furthermore, 4-ethylphenol:4-ethylguaiacol ratios were calculated and show differences between and similarities within varieties. An attempt was made to estimate the precursor quantities originally present in the wines by comparing the results of spiked and unspiked wines but the obtained results were inconclusive. From the results it can be suggested that the grape variety may have an influence on the production of volatile phenols by predetermining the availability of precursors. However, several other factors including vinification and wine aging methods may have an impact on the production of volatile phenols

Survival, metabolism and production of volatile phenols by Dekkera bruxellensis in monovarietal wines

The specific defect caused by the production of volatile phenolic compounds in wines is a major economic concern among wine producers at a global scale. Vinylphenols and, specially, ethylphenols may accumulate in wines imparting undesirable odours and flavours, which may strongly affect the wine quality. The main organisms responsible for the production of these compounds are the yeasts Brettanomyces/Dekkera. Several studies have been carried out on this subject over the last 20 years, but scientific work concerning the relationship between the grape varieties and the production of volatile phenols is extremely scarce. Thus, the overall aim of this work was to characterise the survival and metabolic behaviour of Dekkera bruxellensis in monovarietal wines and to evaluate whether certain grape varieties pose a higher risk to develop volatile phenols. The work started by searching for contaminated wines and by isolating strains of Brettanomyces/Dekkera using selective and differential culture media. The percentage of contaminated wines was 37% and all strains isolated were identified as Dekkera bruxellensis. The results support the view that, among the Brettanomyces/Dekkera yeasts, D. bruxellensis is the main species responsible for the production of volatile phenols in wines. Phenotypic variation was found among the strains studied regarding the tolerance to ethanol. The most tolerant strains were able to grow at 14% v/v ethanol and the wine isolates were, in general, more tolerant than the reference strain D. bruxellensis PYCC 4801. Strains, from the most tolerant group, were selected to perform survival and metabolic studies in real wine conditions. Prior to the metabolic studies, a chemical characterisation of monovarietal red wines from eight selected grape varieties (Cabernet Sauvignon, Syrah, Aragonez, Castelão, Touriga Franca, Touriga Nacional, Trincadeira and Vinhão) was performed, focusing on the volatile phenols and on the respective precursor compounds, both on the free and bond forms. It was found that the precursors exist mostly as esters of tartaric acid (caftaric, coutaric and fertaric acids). The predominant free hydroxycinnamic acid was p-coumaric acid, the highest concentrations being found in Syrah and Touriga Franca and the lowest in Touriga Nacional and Trincadeira. Touriga Nacional exhibited the highest difference between bound and free forms. Twenty two % of the wines analysed presented levels of volatile phenols above the perception threshold, the highest vi values being found in Vinhão and Trincadeira. The results show relevant differences among grape varieties but the availability of precursors on the free form may not be the only factor explaining the potential of wines to develop volatile phenols. The survival of D. bruxellensis in monovarietal wines (Touriga Nacional, Cabernet Sauvignon and Syrah) and the metabolism of hydroxycinnamic acids were evaluated. After the inoculation, yeast culturable populations were reduced to undetectable numbers in all wines, but viability measurement by flow cytometry showed that a significant part of the populations were in a viable-but-not-culturable state (VBNC). VBNC cells were metabolically active, causing the spoilage of the wine, and this physiological state favoured the accumulation of vinylphenols rather than ethylphenols independently of the grape variety. The survival capacity of D. bruxellensis was higher on Touriga Nacional wines than in Cabernet Sauvignon and Syrah. Wine susceptibility to Dekkera may be correlated with the phenolic composition, specifically the bound/free molar ratios of hydroxycinnamic acids. As stated above, Touriga Nacional exhibited the highest difference between bound and free forms and the level of free hydroxycinnamic acids is relatively low in comparison with other grape varieties. Therefore, the documented antimicrobial activity of free hydroxycinnamic acids may be expected to be lower in Touriga Nacional wines. Besides the production of volatile phenols, D. bruxellensis showed esterase activity leading to the increase on the amount of ethyl esters, including ethyl acetate. The fatty acids isovaleric acid and caprylic acid also increased in the inoculated wines. The concentration of monoterpenes analysed increased in the Cabernet Sauvignon wines but not in Touriga Nacional wines. The grape variety effect observed can be related with different compositions of glycosidically bound terpenes subjected to the ß-glycosidase activity of D. bruxellensis.A produção de fenóis voláteis em vinhos tem sérias repercussões económicas nas empresas, pelo que continua a ser fonte de grande preocupação na indústria vínica a nível mundial. A presença de vinilfenóis e, em especial, os etilfenóis acima de determinados valores confere odores e sabores indesejáveis que podem afectar, drasticamente, a qualidade do vinho. Os principais organismos responsáveis pela produção desses compostos são as leveduras Brettanomyces/Dekkera. Diversos estudos foram realizados sobre este assunto nos últimos 20 anos, focando principalmente na origem/fontes de Brettanomyces/Dekkera, nas condições necessárias para o seu desenvolvimento no vinho, nos requisitos nutricionais, em métodos de detecção e identificação e em estratégias para controlar o problema. Mas, trabalho científico sobre a relação entre as castas de uvas e a produção de fenóis voláteis é escasso. Assim, o objetivo geral desta tese foi caracterizar a sobrevivência e o comportamento metabólico de Dekkera bruxellensis em vinhos monovarietais e avaliar se determinadas castas apresentam maior risco de desenvolvimento de fenóis voláteis. O trabalho começou pela pesquisa de vinhos contaminados e pelo isolamento de estirpes de Brettanomyces/Dekkera usando meios de cultura seletivos e diferenciais. A percentagem de vinhos contaminados foi de 37% e todas as estirpes isoladas foram identificadas como Dekkera bruxellensis. Os resultados confirmam que, entre as leveduras Brettanomyces/Dekkera, D. bruxellensis é a principal espécie responsável pela produção de fenóis voláteis nos vinhos. Foi observada variação fenotípica entre as estirpes estudadas quanto à tolerância ao etanol. As mais tolerantes foram capazes de crescer na presença de 14% v/v etanol e, de uma forma geral, os isolados vínicos foram mais tolerantes do que a estirpe de referência D. bruxellensis PYCC 4801. Estirpes do grupo mais tolerante foram selecionadas para realizar estudos metabólicos e de sobrevivência em condições reais de vinho. Antes dos estudos de metabolismo, foi feita a caracterização química de vinhos tintos monovarietais de oito castas selecionadas (Cabernet Sauvignon, Syrah, Aragonez, Castelão, Touriga Franca, Touriga Nacional, Trincadeira e Vinhão), focando nos fenóis voláteis e respectivos compostos precursores, tanto na forma livre como na forma ligada. Verificou-se que viii os precursores existem principalmente como ésteres de ácido tartárico (ácido caftárico, coutárico e fertárico). O ácido hidroxicinâmico predominante foi o ácido p-cumárico, sendo as maiores concentrações encontradas em Syrah e Touriga Franca e as mais baixas em Touriga Nacional e Trincadeira. A casta Touriga Nacional exibiu a maior diferença entre formas livres e ligadas. Vinte e dois % dos vinhos analisados apresentaram níveis de fenóis voláteis acima do limiar de percepção, sendo os valores mais elevados encontrados em Vinhão e Trincadeira. Os resultados mostram diferenças relevantes entre as castas, mas a disponibilidade de precursores na forma livre não é o único fator que explica o potencial dos vinhos para desenvolver fenóis voláteis. A sobrevivência de D. bruxellensis em vinhos monovarietais (Touriga Nacional, Cabernet Sauvignon e Syrah) e o metabolismo dos ácidos hidroxicinâmicos foram avaliados. Após a inoculação, as populações de leveduras cultiváveis foram reduzidas para números indetectáveis em todos os vinhos, mas a determinação da viabilidade por citometria de fluxo mostrou que uma parte significativa das populações estava em no estado viável mas não cultivável (VNC). Células VNC mostraram ser metabolicamente ativas causando a deterioração do vinho, e este estado fisiológico favoreceu a acumulação de vinilfenóis, independentemente da casta. A capacidade de sobrevivência de D. bruxellensis foi maior nos vinhos Touriga Nacional do que em Cabernet Sauvignon e Syrah. A susceptibilidade do vinho a Dekkera pode estar correlacionada com a composição fenólica dos vinhos, especificamente a proporção de ácidos hidroxicinâmicos livres/ligados. Tal como acima referido, a casta Touriga Nacional exibiu a maior diferença entre as formas livres e ligadas e concentração de ácidos hidroxicinâmicos livres é relativamente baixo em comparação com as outras castas. Assim, podemos esperar que a reconhecida atividade antimicrobiana dos ácidos hidroxicinâmicos livres seja menor nos vinhos Touriga Nacional. Além da produção de fenóis voláteis, D. bruxellensis mostrou atividade esterásica levando ao aumento da quantidade de ésteres etílicos, incluindo o acetato de etilo. Os ácidos gordos isovalérico e caprílico também aumentaram nos vinhos inoculados. A concentração de monoterpenos analisados aumentou nos vinhos Cabernet Sauvignon mas não nos vinhos Touriga Nacional. A influência da casta pode estar relacionada com a composição de terpenos ligados a açúcares, sujeitos à atividade ß-glicosidásica de D. bruxellensis

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.info:eu-repo/semantics/acceptedVersio