Article

Enzymatic formation of styrene during wheat beer fermentation is dependent on pitching rate and cinnamic acid content

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Abstract

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reaction processes simultaneously to the decarboxylation of ferulic- and p-cumaric acid to clove-like 4-vinylguaiacol and phenolic 4-vinylphenol by the same PAD1 and FDC1 decarboxylase enzymes. However, the formation of styrene occurs much faster within the first hours of fermentation. In addition, the conversion of cinnamic acid starts immediately after pitching without an adaption of yeast on the new medium. Only after 120 min does the level of transposition decrease. Moreover, high cinnamic acid content in pitching wort, in combination with an open fermentation management, causes faster and higher styrene formation during this period. In contrast to the formation of 4-vinylguaiacol, a correlation between pitching rate and styrene formation during open fermentation could be shown. The resulting time interval between styrene and 4-vinylguaiacol formation provides scope for minimization strategies for styrene, while maintaining the typical wheat beer flavours. Copyright © 2012 The Institute of Brewing & Distilling

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... La fermentación alcohólica se fundamenta en la conversión bioquímica de azúcares como glucosa y/o fructosa, en etanol, proceso llevado a cabo por diversos microorganismos, siendo el más usual la levadura Saccharomyces cerevisiae [1], que como todo ser vivo requiere de nutrientes, los cuales obtiene de los sustratos sobre los cuales crece y desarrolla sus procesos metabólicos. No obstante, los sustratos utilizados por S. cerevisiae como fuente de nutrientes pueden contener otras sustancias que afecten su metabolismo, produciendo una inhibición de su crecimiento y de su producción de etanol [1][2][3][4][5]. ...
... La utilización de una cepa tolerante de S. cerevisiae se basa en su capacidad para metabolizar estos inhibidores y convertirlos en sus derivados menos tóxicos. Por ejemplo, el furfural, o-vainillina y ácido trans-cinámico (Figura 1) pueden ser convertidos en alcohol furfurílico, alcohol o-vainíllico y estireno, respectivamente, por S. cerevisiae [1,2,9,13]. ...
... Un aspecto importante es el comportamiento de los inhibidores metabólicos durante el proceso fermentativo. Diversos estudios han reportado la bioconversión de estos compuestos en sustancias menos nocivas para S. cerevisiae [1,2,9,13], no obstante, es importante conocer las concentraciones residuales de estos inhibidores metabólicos y las tasas de conversión en sus derivados. En el último caso, solamente se ha reportado tasas de bioconversión de furfural por S. cerevisiae [19], y para los demás inhibidores se han postulado hipótesis respecto a cuál sería el destino de estos inhibidores durante el proceso fermentativo [2], o de compuestos análogos como en el caso de vainillina para tratar de explicar la bioconversión de su isómero o-vainillina, con mayor efecto inhibitorio [20]. ...
... The formation of flavour-active volatile phenols 4-vinylguaiacol and 4-vinylphenol, as well as the undesired and carcinogenic styrene, by decarboxylation of their respective precursors (ferulic acid, p-coumaric acid and cinnamic acid) during wort boiling may also have a significant impact on the content of the involved phenolic acids (Langos & Granvogl, 2016;Schwarz, Boitz, & Methner, 2012a). As represented in Fig. 3, the thermal decarboxylation of ferulic acid only occurs during wort boiling and is dependent on temperature, time and ferulic acid concentration (33% and 98% decomposition at 250 • C and 300 • C after) (Cheng, Xu, Liu, Zhao, Xue, & Zhao, 2014). ...
... These enzymes can be found in Saccharomyces cerevisiae strains with Pof + phenotype (Phenolic Off-Flavour Positive) (Langos & Granvogl, 2016). Several authors have clearly demonstrated that the decarboxylation of cinnamic acid, caffeic acid, ferulic acid and p-coumaric acid into styrene, 4-vinylcatechol, 4-vinylguaiacol, 4-vinylphenol, respectively, occurs in higher extension due to enzymatic transformation than due to thermal reaction during wort boiling (Langos & Granvogl, 2016;Richard, Viljanen, & Penttila, 2015;Schwarz et al., 2012a). In fact, a significant reduction of ferulic acid, p-coumaric acid and cinnamic acid was registered after fermentation with a S. cerevisiae strain with normal Pof + activity, while a strain without Pof + activity resulted in higher phenolic acid content after fermentation (Langos & Granvogl, 2016). ...
... The evaluation of styrene formation demonstrated that the enzymatic decarboxylation and cinnamic acid conversion starts immediately after pitching and it is very fast during the first hours of fermentation. The enzymatic conversion was also dependent on the concentration and on fermentation management and temperature (Schwarz et al., 2012a). Recent strategies have explored the production of yeast Pof-variants, allowing to achieve lower levels of phenolic off-flavours and higher phenolic content in beer. ...
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This review provides an overview on the influence of malting and brewing on the overall phenolic content of barley malt and beer. Beer phenolics are mainly originated from barley malt and can be found in free and bound forms, in concentrations up to 50% lower comparing to sweet wort. The use of roasted malts, in combination with proper milling and high mashing temperatures at low pH can lead to a release of bound phenolic forms and increased extraction. New technological strategies such as special yeasts, manipulation of enzymatic activity and dry-hopping may be relevant to improve the phenolic profile of beer and attain phenolic levels with benefits both for beer stability and consumer’s health. As the content of free ferulic acid in beer only accounts up to approximately 15% of total content, further studies should put emphasis on its bound forms in different beer styles and non-alcoholic beers.
... Several studies [12][13][14][15] also reported on the occurrence of styrene in wheat beer, a toxicologically relevant compound [16,17] with a similar molecular structure compared to the desired phenolic aroma compounds. Styrene is classified as "possibly carcinogenic to humans" (group 2B) by the International Agency for Research on Cancer (IARC) [18]. ...
... Nevertheless, the World Health Organisation (WHO) determined a tolerable daily intake (TDI) of 7.7 µg/kg body weight for drinking water [21]. The formation pathway of styrene in beer is similar to the generation of the desired odorants: a thermally, but mainly enzymatically induced decarboxylation during wheat beer brewing, with free cinnamic acid as the respective precursor [11][12][13][14]. The conversion of free cinnamic acid into styrene by Pof + yeast strains of S. cerevisiae was proven to be even faster compared to free ferulic and p-coumaric acid [13], with almost a complete transformation after the whole fermentation process [11]. ...
... The influence of mashing conditions on the release of free phenolic acids as well as a subsequent styrene formation from cinnamic acid during fermentation was investigated in several studies [14,28,29,31,32] suggesting a mitigation of styrene in wheat beer by varying parameters like the pitching rate, fermentation temperature, or fermentation tank (open or closed). In a recent study, it was already shown that the concentrations of free phenolic acids in wheat and especially barley malts depended on parameters of malt production, such as steeping degree, germination temperature, and germination time [33]. ...
Article
Full-text available
2-Methoxy-4-vinylphenol and 4-vinylphenol are important odorants in wheat beer, which are mainly enzymatically formed by decarboxylation during fermentation with the top fermenting yeast Saccharomyces cerevisiae from their respective precursors, the free phenolic acids ferulic acid and p-coumaric acid. However, also the undesired toxicologically relevant styrene can be formed from cinnamic acid by the same mechanism. Micro-brewing trials with specially produced barley and wheat malts were performed, and the concentrations of the free phenolic acids (precursors) and the corresponding vinyl aromatics were quantitated in the malts and the intermediates of beer production (unboiled wort, cast wort, and green beer) using stable isotopically labelled internal standards. Thereby, 100% wheat or barley malts produced with either high or low malting parameters (steeping degree, germination temperature, and germination time) were used for brewing. Furthermore, the influence of two different yeasts, Saccharomyces cerevisiae strain W68 (top fermenting) and Saccharomyces carlsbergensis strain W34/78 (bottom fermenting), on the amounts of the phenolic acids and the corresponding vinyl aromatics was investigated. Results indicated that modifications in barley malting parameters did not have a mitigating influence on the styrene concentration in beer. However, using wheat malt produced with low malting parameters for brewing revealed a reduced styrene amount by about 30% compared to the brew produced with wheat malt obtained by high malting parameters. In addition, the lowered styrene beer did still fulfil the consumer´s expectation in regard to its overall aroma.
... Several studies by Langos et al. (1,(9)(10)(11), Langos (12), and Schwarz et al. (8,(13)(14)(15)(16) examined the influence of different process steps of brewing on the release of styrene and the desired vinyl aromatics. It was shown that thermal process steps, like mashing, wort boiling, and pasteurization, have little to no contribution to the overall styrene concentration caused by thermal decarboxylation. ...
... Therefore, different ratios between styrene, 4VP, and 2M4VP within the remaining set of pale wheat beer samples in Table 2 are influenced by other factors, for example, barley and wheat varieties used (23,24), malting conditions applied (12), malt ratio used (8,25), or mashing conditions applied (13,26). All of them may affect the input of free phenolic acids into the brewing process as well as the overall fermentation management (14,15,27). This explains why the calculated Pearson correlation coefficients of a dataset including only pale wheat beers showed no significant correlations. ...
... For the latter, different methods are applied (e.g., thermal dealcoholization, reducing the alcohol content by evaporation, or reverse osmosis, removing the alcohol via a semipermeable membrane). Schwarz et al. (15) proved a nearly complete conversion of cinnamic acid into styrene after 2 h after starting the fermentation by addition of Pof +/− active yeasts. Therefore, it is expected that a stopped fermentation has no effect on the styrene concentration but does reduce the content of the desired vinyl aromatics as they reach their maximum of release after 3 to 6 days, depending on the fermentation temperature (14). ...
... Steele et al. (1994) determined styrene in 8 of 12 selected food types, including cinnamon, beef, coffee beans, peanuts, wheat, oats, pecans, strawberries, and peaches, but this compound was not found in tomatoes, chicken and milk. Enzymatic formation of styrene is also possible (Schwarz et al., 2012) because pollen is produced by bees with the addition of their saliva. ...
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Characterization of volatile compounds composition and evaluation of antioxidant properties of bee pollen collected in Lithuania is presented in the paper, which is the first study on pollen of Lithuanian origin to our knowledge. Three polyfloral pollen samples collected by honey bees were analyzed. Characterization of volatile compounds was performed using solid phase microextraction (SPME) and GC-MS. Styrene was predominant in all samples contributing 19.6-27.0 mass %. Sample A distinguished by a high amount of limonene, 9.0 mass %, sample B differed from the rest by a high content of hexanal, 9.3 mass %, and nonanal, 12.3 mass %, while sample C showed the highest content of 1-tridecene, 43.3 mass %. Screening of antioxidant properties was carried out by spectrophotometric methods and liquid chromatography coupled with a post-column 1,1- diphenyl-2-picrylhydrazyl (DPPH) reaction detector and electrochemical detector. Total phenolic and flavonoid contents together with radical scavenging activity in the free radical (DPPH) model system were evaluated in the tested samples. Total phenolic content varied from 24.4 mg g-1 to 38.9 mg g-1, total flavonoid content was in the range of 7.3-10.0 mg g-1 and radical scavenging activity was found between 30.7 mg g-1 and 34.9 mg g-1, all data are expressed in rutin equivalents. To process the collected data statistically and classify the bee pollen samples to clusters according to their volatile composition and antioxidant activity, principal component analysis, hierarchical cluster analysis and non-linear discriminant analysis were applied.
... Furan has been classified as 'possibly carcinogenic to humans' (group 2B) by the IARC (1995). Styrene (a benzene derivative) is formed by the thermal decarboxylation of cinnamic acid during beer wort boiling or by enzymatic decarboxylation during alcoholic fermentation (Schwarz et al. 2012) and has also been included in group 2B by the IARC (2002), on the basis of limited evidence of its carcinogenicity in animal tests, supported by an extensive set of other relevant data, including biomarkers of exposure and effect. ...
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This study investigates the effect of treatment with activated carbon and other adsorbents on the chemical composition and organoleptics of a barley malt-based agricultural distillate. Contact with activated carbon is one of the methods by which the quality of raw distillates and spirit beverages can be improved. Samples placed in contact with 1 g activated carbon (SpiritFerm) per 100 ml distillate with ethanol content of 50% v v(-1) for 1 h showed the largest reductions in the concentrations of most volatile compounds (aldehydes, alcohols, esters). Increasing the dose of adsorbent to over 1 g 100 ml(-1) did not improve the purity of the agricultural distillate significantly. Of the tested compounds, acetaldehyde and methanol showed the lowest adsorption on activated carbon. The lowest concentrations of these congeners (expressed in mg l(-1) alcohol 100% v v(-1)) were measured in solutions with ethanol contents of 70-80% v v(-1), while solutions with an alcoholic strength by volume of 40% did not show statistically significant decreases in these compounds in relation the control sample. The reductions in volatile compounds were compared with those for other adsorbents based on silica or activated carbon and silica. An interesting alternative to activated carbon was found to be an adsorbent prepared from activated carbon and silica (Spiricol). Treatment with this adsorbent produced distillate with the lowest concentrations of acetaldehyde and isovaleraldehyde, and led to the greatest improvement in its organoleptics.
... La especie Saccharomyces cerevisiae ha sido evaluada en diferentes trabajos previos, mostrando resistencia debido a su capacidad para secretar enzimas con impacto positivo en la bioconversión de estos inhibidores en metabolitos menos tóxicos. Se ha reportado que el furfural, o-vainillina, glicolaldehído o ácido cinámico pueden ser metabolizados y convertidos por S. cerevisiae en alcohol furfurílico, alcohol o-vainíllico, etilenglicol y estireno, respectivamente [10,11,[14][15][16]. ...
... The styrene concentration in milled olive pulp increased significantly during storage at ambient temperature, but did not change significantly when stored in the refrigerator, suggesting that styrene in olives is a product of metabolism (Biedermann, Grob, and Morchio 1995). Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation of wheat beer, which can contain concentrations up to 25 µg/L (Schwarz, Boitz, and Methner 2012). Use of specialized yeast strains might reduce styrene concentrations in wheat beer without loss of desired aroma (Langos, Gastl, and Granvogl 2017;Langos and Granvogl 2016). ...
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The potential chronic health risks of occupational and environmental exposure to styrene were evaluated to update health hazard and exposure information developed since the Harvard Center for Risk Analysis risk assessment for styrene was performed in 2002. The updated hazard assessment of styrene’s health effects indicates human cancers and ototoxicity remain potential concerns. However, mechanistic research on mouse lung tumors demonstrates these tumors are mouse-specific and of low relevance to human cancer risk. The updated toxicity database supports toxicity reference levels of 20 ppm (equates to 400 mg urinary metabolites mandelic acid + phenylglyoxylic acid/g creatinine) for worker inhalation exposure and 3.7 ppm and 2.5 mg/kg bw/day, respectively, for general population inhalation and oral exposure. No cancer risk value estimates are proposed given the established lack of relevance of mouse lung tumors and inconsistent epidemiology evidence. The updated exposure assessment supports inhalation and ingestion routes as important. The updated risk assessment found estimated risks within acceptable ranges for all age groups of the general population and workers with occupational exposures in non-fiber-reinforced polymer composites industries and fiber-reinforced polymer composites (FRP) workers using closed-mold operations or open-mold operations with respiratory protection. Only FRP workers using open-mold operations not using respiratory protection have risk exceedances for styrene and should be considered for risk management measures. In addition, given the reported interaction of styrene exposure with noise, noise reduction to sustain levels below 85 dB(A) needs be in place.
... Several studies on the reduction of styrene in wheat beer were examined by Langos and Granvogl [11], Langos et al. [23,24], Schwarz and Methner [25], and Schwarz et al. [26][27][28][29], mainly focusing on the impact of brewing steps after malting, such as mashing, wort boiling, and fermentation. Thereby, found styrene mitigation strategies within these processing steps were mostly accompanied by a significant reduction of the desired vinyl aromatics and, therefore, with the risk to lose the characteristic wheat beer aroma. ...
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Full-text available
Aiming at the mitigation of the toxicologically relevant styrene formed during wheat beer brewing, different malting parameters, such as steeping temperature, germination temperature, withering and kilning temperatures applied during kiln-drying, and aeration rate, were evaluated for their suitability to reduce the content of cinnamic acid, the precursor of styrene, in malts of barley and wheat, responsible for the input of the undesired precursor into the brewing process. According to the results of the present study, higher steeping temperatures, higher germination temperatures, lower aeration rates, and lower withering temperatures during malting are beneficial for the overall reduction of cinnamic acid in wort produced with barley and wheat malts. Thereby, the withering temperature showed the highest impact among the investigated parameters, able to reduce the soluble cinnamic acid content in wort by up to 72%, followed by the germination temperature in combination with the aeration rate and the steeping temperature with reduction capacities of 52 and 16%, respectively. Additionally, a kilning temperature of 200 °C led to the absence of enzyme activities in dark malts, which might also be the main reason for the low phenolic acid contents found in the corresponding wort, finally causing the low concentrations of styrene but also to a certain extent of desired vinyl aromatics in dark wheat beers.
... Styrene is another VOC reported in apple brandy and cider, with odour threshold values ranging between 3.6 to 80 µg L −1 [30], and in apple fruits [31]. The formation of this VOC may be because high cinnamic acid content and yeast pitching rate, in combination with open fermentation management, cause quick and increased styrene formation during fermentation, as was previously reported for wheat beer [32]. Thus, styrene may be used as an important indicator to monitor the cider-making process (as well as in beers) and management with food authentication purposes. ...
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... The prominent yeast enzyme FDC1 belongs to the class of prFMN dependent decarboxylases and requires a cofactor regenerating system. It shows broader substrate specificities resulting in the formation of the putative carcinogen styrene from cinnamic acid, as it is present in top-fermented beverages, such as wheat beer, and is thus not a suitable candidate [12,13]. Cofactor independent decarboxylases including bacterial, plant and ascomycetous FADs, require a mandatory hydroxyl group in para position of the substrate and are not capable of producing styrene. ...
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... In our study, its relative content was only 1% of the total volatiles of bee pollen, which may indicate that bee pollen from Poland may be less contaminated. According to the available literature, styrene in bee pollen can come from plastic bags [27], but it may also exist naturally or could be produced during enzymatic synthesis since bee pollen is produced by bees with the use of their saliva [28]. ...
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The screening of styrene concentrations during different stages of wheat beer production shows that in opposite to thermal processes like mashing and wort boiling the primary and secondary fermentation contribute most to the styrene content in final beers. Therefore, the enzymatic decarboxylation of cinnamic acid by brewers' yeast is the predominant kind of reaction. Styrene concentrations in bottled beer were up to 25 ppb. In addition, the results of the screening were compared with the precursor concentration of cinnamic acid in the corresponding pitching wort. A linear correlation could not be found. Moreover, the Influence of various manufacturing considerations like the kind of fermentation or the ratio of wheat grist load is shown. For example, the cinnamic acid content in wort decreases with an increasing amount of wheat malt.
Article
Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof+ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pofδ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.
Article
In this study the ideal mashing-in conditions for wheat beer were examined. The parameters concerning temperature, pH value, and time should guarantee an optimal activity of arabinoxylan- and protein-degrading enzymes and thus a high release of ferulic acid and p-coumaric acid as well as amino acids. These phenolic acids are precursors of the flavorand aroma-active volatile phenols 4-vinylguiacol and 4-vinylphenol. Together with higher alcohols and esters, e.g., phenyl ethanol and isoamyl acetate, formed among others by amino acids, they make a decisive contribution to the typical aroma and taste profile of wheat beer. In addition to the maximized release of ferulic, p-coumaric acid, and amino acids, the investigated mashing-in conditions should reduce the amount of cinnamic acid. This phenolic acid, which is naturally prevalent in cereals, is converted by thermal and enzymatic decarboxylation into the harmful substance styrene during the brewing process. The studied parameters, especially the mashing-in pH, afford the realization of the set targets. Besides the determination of the effects of different mashing-in parameters, various colored malts and raw materials were investigated for their contents of phenolic acids and amino acids.
Article
Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reactions proceed in parallel to the decarboxylation of ferulic- and p-cumaric acid to 4-vinylguaiacol and 4-vinylphenol by the same decarboxylase enzyme. However, the formation of styrene occurs much faster and all available cinnamic acid in wort was converted completely within a few hours. Moreover, the comparison of various manufacturing parameters shows that a higher fermentation temperature of 25°C compared to 16°C and an open fermentation management lead to a rapid decrease of styrene. This allows minimising the content of styrene in beer while maintaining the typical wheat beer flavours.
Article
Styrene levels in 12 commodities were determined. The foods tested were wheat, oats, peanuts, pecans, coffee beans, tomatoes; peaches, strawberries, cinnamon, beef, chicken, and milk. The samples were collected in a manner that avoided contact with styrene or any type of plastic. Analytical measurements were performed using a dynamic heated headspace purge-and-trap extraction technique followed by quantification by selected ion monitoring capillary gas chromatography/mass spectrometry. Method blanks, duplicate samples, and samples fortified with benzene and styrene were used to assess method performance. The limit of detection of the method varied between food types but was generally less than 2 ng/g for styrene. The highest measured concentrations of styrene were found in cinnamon (169-39 200 ng/g). Styrene concentrations in beef samples ranged from 5.25 to 7.85 ng/g and in coffee beans from 1.57 to 7.85 ng/g. Wheat, pecans, oats, strawberries, and peaches showed no styrene concentrations greater than 3 ng/g. No detectable styrene was found in tomatoes, milk, or chicken.
Article
The stereochemistry of the decarboxylation of 3,4-dimethoxycinnamic acids by Saccharomyces cerevisiae and the enzymatic specificity with respect to the substrate structure were studied. This reaction proceeds with retention of configuration at the side-chain double bond as well as enzymatic specificity for the (E) configuration. The influence of substituents in the α and β positions was also studied. Hypotheses on the reaction mechanism were proposed.
Article
Phenole, Phenolther und aromatische Kohlenwasserstoffe sind Bestandteile vieler Lebensmittel und oft typische Aromastoffe beispielsweise von Rst- und Rucherprodukten. Sie haben auch toxikologische Bedeutung. In Modellversuchen wurde ihre Bildung durch thermische Fragmentierung von Zimtsure, p-Cumarsure, Ferulasure und Sinapinsure eingehend untersucht. Die entstandenen Verbindungen wurden gaschromatographisch bestimmt und massenspektrometrisch bzw. infrarotspektroskopisch charakterisiert. Produktverteilung und Massenspektren sind angegeben. In analoger Weise wurde Gerste gerstet und die erzeugten Komponenten identifiziert. Zur Fraktionierung wurden dabei Kieselgel-Sulenchromatographie und prparative Gaschromatographie eingesetzt.Phenolic compounds and aromatic hydrocarbons are components in many foods and often typical flavoring substances for example of roasted and smoked products. They are also of toxicological importance. By means of model reactions we have investigated their formation by thermal fragmentation of cinnamic, p-coumaric, ferulic and sinapic acids. The products of these reactions were determined by gas chromatography and identified by mass spectrometry or infrared spectroscopy. Product ratio and mass spectra are given. Barley was roasted in a similar manner, and the components formed were identified. Column chromatography and preparative gas chromatography were used for preliminary separation.
Article
In this study, the effects of mashing variables such as mashing-in temperature, time and pH, mash thickness, grist coarseness and composition, and stirring regime on the release of ferulic acid were examined. Ferulic acid is a precursor for the formation of flavour-active volatile phenols and a potent natural antioxidant in beer. Given one barley malt variety, the multitude of choice in setting various process parameters and adding brewery adjuncts during brewhouse operations can give rise to worts with widely varying ferulic acid levels. A clear difference in temperature- and pH-dependence between the release of the water-extracted and the enzymatically hydrolyzed fraction was found. The T,t-dependencies of arabinoxylan-degrading enzyme activities were correlated with ferulic acid release during mashing. Results from laboratory-scale mashing experiments were validated with those from a pilot-scale (5 h) wort production process. Enhancing the enzymatic release of phenolic flavour precursors from bound forms during mashing can greatly enhance the phenolic aroma potential of wort. Optimising this precursor release during mashing may be a means for controlling final volatile phenol levels in beer.
Article
Inhibition of yeast function by ethanol and by high substrate concentrations is well recognized and, to a limited extent, quantified. The role of carbon dioxide in affecting yeast metabolism (particularly growth processes) is not clear although inhibition is generally found at moderate to high concentrations of the dissolved gas. A similar situation exists with other microorganisms and with other fermentation systems. An understanding of the role of carbon dioxide, and particularly of its inhibitory effects on enzyme action and membrane function, is required if the observed global inhibition of yeasts and other fermentation systems is to be partitioned to its appropriate causes.
Article
The volatile phenols, to which Saccharomyces cerevisiae converts from phenylacrylic acids including ferulic acid, p-coumaric acid, and cinnamic acid, generate off-flavors in alcoholic beverages such as beer and wine. Using gene disruptants, transformants and cell-free extracts of these strains, we have verified that the adjacent PAD1 (phenylacrylic acid decarboxylase, YDR538W) and FDC1 (ferulic acid decarboxylase, YDR539W) genes are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae. Pad1p and Fdc1p are homologous with UbiX and UbiD, respectively, in the ubiquinone synthetic pathway of Escherichia coli. However, ubiquinone was detected quantitatively in all of the yeast single-deletion mutants, Delta pad1, Delta fdc1, and double-deletion mutant, Delta pad1 Delta fdc1.
Article
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Article
Styrene is metabolized to styrene oxide, a direct-acting mutagen and carcinogen. Styrene oxide reacts with DNA mainly at the N-7 position in guanine, but also at other sites and with other bases. Substitution occurs at both the alpha- and beta-positions of the styrene molecule. Experiments with radiolabeled styrene and styrene oxide demonstrate that both have a low level of DNA binding activity in experimental animals. 32P-Postlabeling studies have demonstrated the potential of the technique to detect styrene-DNA adducts. Styrene oxide alkylates several nucleophilic sites in proteins, particularly cysteine sulfydryl, histidine imidazole, lysine amino, aspartic, and glutamic carboxylic groups, and the N-terminal position. In experimental animals, styrene oxide treatment results in cysteine adducts in hemoglobin and albumin, valine adducts in hemoglobin, and carboxylic acid adducts in hemoglobin. The extent of alkylation is low compared with that produced by ethylene oxide. The available evidence indicates, therefore, that styrene and styrene oxide have low DNA and protein binding activities in vivo. There is preliminary evidence for the presence of DNA adducts and for adducts in hemoglobin and albumin in blood cells of styrene-exposed workers. Nevertheless, the applicability and sensitivity of DNA and protein adduct detection methods for monitoring human exposure to styrene remain to be determined.
Article
Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n-1)-phosphate with chain length from 2 to 8 bases. Alkylated and nonalkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O6- and N2-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable.
Article
Epoxides react at various nucleophilic sites in macromolecules such as haemoglobin and DNA. To study the reaction rate constants of ethylene oxide (EO), propylene oxide (PO) and styrene 7,8-oxide (SO) towards two of these positions, i.e., the N-terminal valine in haemoglobin and N-7-guanine in DNA was the central aim of this investigation. These two reactive sites are the most studied haemoglobin and DNA adducts, respectively. Further attention, therefore, was also paid to the applicability in vivo of the in vitro determined reaction constants. The determination of the second-order rate constants between EO and PO and N-terminal valine in Hb [2.7 l (mol Hb h)-1 and 1.0 l (mol Hb h)-1, respectively] were consistent with the literature values. The constants for the reaction with N-7-guanine [16x10(-3) l (mol DNA nucleotide h)-1 and 7. 7x10(-3) l (mol DNA nucleotide h)-1, respectively] were lower than previously published values, probably due to differences in the methodology used. The use of the in vitro obtained values to model the in vivo situation lead to a consistent picture for EO and PO. In contrast, for SO the in vitro ratio between the adduct formation on N-terminal valine [1.5 l (mol Hb h)-1] and N-7-guanine [0.71x10(-3) l (mol DNA nucleotide h)-1] was about two orders of magnitude greater than for the in vivo situation. This was probably due to a lower than expected reactivity of SO towards N-terminal valine in vivo. Further research is needed to elucidate whether the use of SO in vitro, contrasting with the in vivo experiments in which SO was metabolically formed from styrene, could entail an explanation for this discrepancy. Concerning the methodological part, the use of dipeptide standards to replace the alkylated globins as standard lead to an improvement of the method. Especially the commercial availability of the standards, their stability and accurately known adduct content will make them to the standards of choice in the future.
Article
In the present studies, human exposure to styrene and to ethylbenzene (EB) is assessed on the basis of literature data. Total styrene and total EB exposure result from inhalation and from food intake. Styrene and EB inhaled represent the greatest proportion of the total intake. Styrene and EB content in food is mainly caused by migration from polymer packaging material. The daily styrene exposure is estimated to range from 18.2 to 55.2 microg/person, corresponding to an annual exposure of 6.7 to 20.2 mg/person. The daily EB exposure is estimated to be about 130 microg/person, corresponding to an annual exposure of 46 mg/person. Cigarette smoking is another important factor for styrene and EB intake by smokers.
Article
The formation of 4-vinylguaiacol, guaiacol, and phenol during coffee roasting was monitored in real-time, using resonance enhanced multiphoton ionization and time-of-flight mass spectrometry. A model is proposed, based on two connected reaction channels. One channel, termed the "low activation energy" channel, consists of ester hydrolysis of 5-FQA followed by decarboxylation of the ferulic acid to form 4-vinylguaiacol, and finally polymerization at the vinyl group to form partly insoluble polymers (coffee melanoidins). The second "high activation energy" channel opens up once the beans have reached higher temperatures. It leads to formation of guaiacol, via oxidation of 4-vinylguaiacol, and subsequently to phenol and other phenolic VOCs. This work aims at developing strategies to modify the composition of coffee flavor compounds based on the time-temperature history during roasting.
Article
The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process. Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt. To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt. Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process. During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling. Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype. In beer made with barley malt, ferulic acid was mainly released during the brewing process. Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers. As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.
Article
The spoilage yeast Saccharomyces cerevisiae degraded the food preservative sorbic acid (2,4-hexadienoic acid) to a volatile hydrocarbon, identified by gas chromatography mass spectrometry as 1,3-pentadiene. The gene responsible was identified as PAD1, previously associated with the decarboxylation of the aromatic carboxylic acids cinnamic acid, ferulic acid, and coumaric acid to styrene, 4-vinylguaiacol, and 4-vinylphenol, respectively. The loss of PAD1 resulted in the simultaneous loss of decarboxylation activity against both sorbic and cinnamic acids. Pad1p is therefore an unusual decarboxylase capable of accepting both aromatic and aliphatic carboxylic acids as substrates. All members of the Saccharomyces genus (sensu stricto) were found to decarboxylate both sorbic and cinnamic acids. PAD1 homologues and decarboxylation activity were found also in Candida albicans, Candida dubliniensis, Debaryomyces hansenii, and Pichia anomala. The decarboxylation of sorbic acid was assessed as a possible mechanism of resistance in spoilage yeasts. The decarboxylation of either sorbic or cinnamic acid was not detected for Zygosaccharomyces, Kazachstania (Saccharomyces sensu lato), Zygotorulaspora, or Torulaspora, the genera containing the most notorious spoilage yeasts. Scatter plots showed no correlation between the extent of sorbic acid decarboxylation and resistance to sorbic acid in spoilage yeasts. Inhibitory concentrations of sorbic acid were almost identical for S. cerevisiae wild-type and Δpad1 strains. We concluded that Pad1p-mediated sorbic acid decarboxylation did not constitute a significant mechanism of resistance to weak-acid preservatives by spoilage yeasts, even if the decarboxylation contributed to spoilage through the generation of unpleasant odors.
Article
Volatile phenols have long been recognized as important flavor contributors to the aroma of various alcoholic beverages. The two main flavor-active volatile phenols in beer are 4-vinylguaiacol and 4-vinylphenol. They are the decarboxylation products of the precursors ferulic acid and p-coumaric acid, respectively, which are released during the brewing process, mainly from malt. In this study, the variability in the release of free and ester-bound hydroxycinnamic acids from nine malted barley ( Hordeum vulgare L.) varieties during wort production was investigated. A large variability between different barley malts and their corresponding worts was observed. Differences were also found between free ferulic acid levels from identical malt varieties originating from different malt houses. During mashing, free hydroxycinnamic acids in wort are both water-extracted and enzymatically released by cinnamoyl esterase activity. Esterase activities clearly differ between different barley malt varieties. Multiple linear regression analysis showed that the release of ferulic acid during mashing did not depend only on the barley malt esterase activity but also on the amount of ester-bound ferulic acid initially present in the wort and on its endoxylanase activity. The study demonstrates the importance of selecting a suitable malt variety as the first means of controlling the final volatile phenol levels in beer.
Biersorten und ihre Besonderheiten
  • W Kunze
Kunze, W. (2007) Biersorten und ihre Besonderheiten, in Technologie Brauer & Mälzer (Kunze, W. Ed.), 9th ed., pp. 881-883, VLB-Berlin, Berlin.
Volatile phenols in beer: formation of 4-vinylguaiacol during wort fermentation and its fate during beer aging Proc
  • N Vanbeneden
Vanbeneden, N. (2009) Volatile phenols in beer: formation of 4-vinylguaiacol during wort fermentation and its fate during beer aging. Proc. Eur. Brew. Conv. Congr., Hamburg, Lecture 24.
Hefeweizenbier -taste spectrum and technology
  • W Back
  • C Diener
  • B Sacher
Back, W., Diener, C., and Sacher, B. (2000) Hefeweizenbier -taste spectrum and technology. Brauwelt Int., 18, 112-119.
Obergärigen Biere und Besonderheiten der Obergärung
  • G Annemüller
  • H J Manger
Annemüller, G. and Manger, H.J. (2009) Obergärigen Biere und Besonderheiten der Obergärung, in Gärung und Reifung des Bieres (Annemüller, G. and Manger, H.J. Eds), 1st ed., pp. 411-427, VLB-Berlin, Berlin.
Abriß der Bierbrauerei
  • L. Narziß
Gärung und Reifung des Bieres
  • G. Annemüller
  • H.J. Manger
and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae
  • N Mukai
  • K Masaki
  • T Fujii
  • M Kawamukai
  • H Lefuji
Mukai, N., Masaki, K., Fujii, T. Kawamukai, M., and Lefuji, H. (2010) PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. J. Biosci. Bioeng., 109(6), 564-569.
  • K J Schwarz
  • L Boitz
  • F.-J Methner
Schwarz, K.J., Boitz, L., and Methner, F.-J. (2012) Release of phenolic acids and amino acids during mashing dependent on temperature, pH, time and raw materials. http://dx.doi.org/10.1094/ASBCJ-2012-0612-01, in press.
Hefeweizenbier - taste spectrum and technology
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