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Conversion of ferulic acid to 4-vinyl guaiacol by yeasts isolated from unpasteurised apple juice
Journal of The Science of Food and Agriculture
Abstract
Yeasts were isolated from unpasteurised apple juice on potato dextrose agar supplemented with tartaric acid and on malt extract agar containing chloramphenicol. Isolates were screened for feruloyl esterase and ferulic acid decarboxylase activities. Strains of Rhodotorula rubra, R minuta and Candida lambica produced both enzymes. Ferulic acid decarboxylase activity, resulting in the formation of the off-flavour 4-vinyl guaiacol, was highest for the C lambica isolate. This decarboxylase activity was associated with the washed resting cells. The crude enzyme showed temperature and pH optima of 35°C and 6.5, respectively.© 1999 Society of Chemical Industry
... Later-stage 4-VG production suggests that decarboxylase activity (FDC) in R mucilaginosa UFMG-CM-Y3647 could be affected not only by pH but also according to microorganism growth stage, as resting cells are commonly used to determine this enzymatic activity. 23,26,28,37,60 After pH definition, we tested a higher aeration condition in order to evaluate the effect of oxygen concentration on VA production. Figure 5c show that higher aeration rate did not improved VA concentration, as maximum metabolite production was Both biomass concentration and VA productivity, however, were significantly increased, with VA productivity of 40.82 ± 0.57 mg/ (LÁhr), 19% higher than for 0.5 vvm, while biomass final concentration increased from 6.52 ± 0.35 to 10.27 ± 0.71 g/L. ...
... 64,65 Under slightly neutral pH ranges (b), which could be free or con- including Rhodotorula species. 35,37 We investigated the ability of R mucilaginosa UFMG-CM-Y3647 ...
Nine yeast strains isolated from Latin American biodiversity were screened for ferulic acid (FA) consumption and conversion into aroma compounds such as vanillin, vanillic acid (VA), and 4‐vinylguaiacol (VG). Selected strains (Rhodotorula mucilaginosa UFMG‐CM‐Y3647, UFMG‐CM‐Y2190, UFMG‐CM‐Y665) were evaluated in flask experiments to investigate the influence of the pH media on bioconversion and a two‐step process was conducted to maximize the metabolites production. The effect of pH was found to be significantly important for FA bioconversion, as acidic conditions (pH < 6.0) improved VA accumulation, with highest production of 1.14 ± 0.02 and 1.25 ± 0.03 g/L shown by UFMG‐CM‐Y3647 and UFMG‐CM‐Y2190, respectively. The two‐step process favored 4‐VG production for most strains, being UFMG‐CM‐Y2190 the best producer, its cultures reaching 1.63 ± 0.09 g/L after 55 hr, showing a productivity of 29.59 ± 1.55 mg/(L·hr), as glucose affected the metabolites pool and redirected yeast metabolism. R mucilaginosa UFMG‐CM‐Y3647 was selected for scaled‐up cultivations in a 2‐L bioreactor, where pH‐controlled pH 5.5 and aeration of 2.5 vvm was found to be the best condition to improve VA productivity, attaining final concentrations of 1.20 ± 0.02 g/L⁻¹ (78% molar yield) and a productivity of 40.82 ± 0.57 mg/(L·hr).
... Biodegradation is a major mechanism for eliminating environmental pollutants and has been studied widely in recent years (Rodríguez et al., 2004;Peng et al., 2013). Moreover, various fungi (Baqueiro-Peña et al., 2010;Donaghy et al., 1999) and bacteria (Civolani et al., 2000) capable of degrading ferulic acid have also been isolated and studied (Mathew and Abraham, 2006). In this work, the ability of P. liquidambari to degrade a model pollutant, ferulic acid, commonly present in agro-industrial effluents, was investigated. ...
... From the perspective of biochemical reactions, the pathways mentioned above need the participation of decarboxylase, oxidoreductase and oxygenase. Donaghy et al. (1999) noted that the initial step of ferulic acid catabolism was catalyzed by FADase in fungi. Promputtha et al. (2010) demonstrated that endophytic fungi could produce extracellular oxidases including laccase, MnP and LiP, which are involved in the degradation of various phenolics. ...
Biodegradation of ferulic acid, by an endophytic fungus called Phomopsis liquidambari was investigated in this study. This strain can use ferulic acid as the sole carbon for growth. Both in mineral salt medium and in soil, more than 97% of added ferulic acid was degraded within 48 h. The metabolites were identified and quantified using GC–MS and HPLC–MS. Ferulic acid was first decarboxylated to 4-vinyl guaiacol and then oxidized to vanillin and vanillic acid, followed by demethylation to protocatechuic acid, which was further degraded through the β-ketoadipate pathway. During degradation, ferulic acid decarboxylase, laccase and protocatechuate 3,4-dioxygenase activities and their gene transcription levels were significantly affected by the variation of substrate and product concentrations. Moreover, ferulic acid degradation was determined to some extent by P. liquidambari laccase. This study is the first report of an endophytic fungus that has a great potential for practical application in ferulic acid-contaminated environments.
... It might be responsible for the smoky phenolic odour note, which is more intense in the Robusta coffee brew (Semmelroch et al., 1996). Vinylphenols have been reported as potential off-flavour compounds in other non-alcoholic drinks like apple juice (Donaghy et al., 1999) and orange juice (Fallico et al., 1996; Lee et al., 1990; Marcotte et al., 1998; Naim et al., 1993; Peleg et al., 1992; Rouseff et al., 1992; Walsh et al., 1997) where they impart an old or rotten fruit aroma. They also contribute to the aroma of various other alcoholic drinks like whisky (Lee et al., 2000; van Beek et al., 2000) and sherry (Dominguez et al., 2002; Tressl et al., 1976 ). ...
... The production of 4VP was detected only in the beginning of the growth phase in low amounts because it was rapidly reduced. 4EP production was observed only after complete fermentation of the grape juices byChatonnet et al., 1992; Donaghy et al., 1999; Mathew et al., 2007; Rosazza et al., 1995; Shinohara et al., 2000; Suezawa et al., 2007). Certain Candida spp. ...
De meeste vluchtige fenolen in bier zijn afkomstig van de grondstoffen die gebruikt worden tijdens het brouwproces. De aroma-actieve fenolen 4-vinylfenol (4VP) en 4-vinylguaiacol (4VG) worden echter hoofdzakelijk gevormd tijdens de fermentatie door brouwersgist. De aanwezigheid van deze componenten is ongewenst in pilsbier. Hierdoor werden ze historische beschouwd als off-flavours . Ze maken echter essentieel deel uit van het karakteristieke aroma van vele blonde speciaalbieren, witbieren en andere tarwebieren waar ze een kruidig of gerookt aroma aan de betreffende bieren verlenen. De laatste jaren worden deze smaakcomponenten in toenemende mate prominent aangetroffen in het aroma van Belgische speciaalbieren. Alhoewel sommige brouwers de aanwezigheid van deze componenten duidelijk wensen in (sommige van) hun bieren, brengen ze het aromaprofiel van vele andere bieren, door hun soms overheersende aroma, uit balans. Er is weinig geweten over de dynamiek van de vrijzetting van hun precursoren (de fenolzuren p -coumarinezuur en ferulinezuur) tijdens het brouwproces en de interactieve rol die de mouteigen enzymen hierbij kunnen spelen. Karakterisering van de betrokken enzymen en onderzoek naar de invloed van de procesparameters tijdens de bierproductie op de vrijstelling van de precursoren en de daaropvolgende omzetting tot vluchtige fenolen is nodig om de brouwers toe te laten de gehaltes aan fenolzuren en vluchtige fenolen te beheersen afhankelijk van de beoogde bierstijl en om een constante batch-to-batch kwaliteit te kunnen afleveren. Vooreerst werd een analytische methode (RP-HPLC-ECD) op punt gesteld en gevalideerd die gebruikt kan worden voor de bepaling van deze componenten en hun precursoren in wort en bier. Deze methode werd gebruikt voor een grootschalige analyse van Belgische speciaalbieren om het voorkomen van vluchtige fenolen en hun precursoren in verschillende bierstijlen in kaart te brengen. Geur- en smaakdrempels werden bepaald in water en in verschillende biermedia om de impact van deze aromacomponenten op het bieraroma te bepalen. Vervolgens werd de evolutie van fenolzuren en vluchtige fenolen opgevolgd tijdens het volledige bierproductieproces om realistische controlepunten te identificeren. Er werd een grote variabiliteit in het fenolzuurgehalte tussen verschillende moutvariëteiten gevonden. Ook werden verschillen aangetroffen tussen gelijke moutvariëteiten afkomstig van verschillende mouterijen. Dit wijst op het belang van de keuze van een gepaste moutvariëteit wanneer men het uiteindelijk gehalte aan vluchtige fenolen in bier wenst te beheersen. De esterase- en endoxylanase-activiteit van de mout alsmede het gehalte aan estergebonden fenolzuren in wort werden geïdentificeerd als verklarende factoren voor het finale gehalte aan vrije fenolzuren in wort. Eveneens na de keuze van de gepaste moutvariëteit is er nog ruimte voor de beheersing van de uiteindelijke fenolzuurconcentratie in wort. Doordat fenolzuren deels worden vrijgezet door een enzymatische reactie tijdens het brouwen, kan men door variatie van allerhande procesparameters in de brouwzaal, het gehalte aan fenolzuren in bier beïnvloeden.
Vrije fenolzuren in wort zijn beschikbaar voor decarboxylatie tot aroma-actieve fenolen. Deze decarboxylatie kan zowel thermisch als enzymatisch geïnitieerd worden. Voor de soms hoge gehaltes aan vluchtige fenolen die vaak aangetroffen worden in blonde speciaalbieren, volstaat thermische decarboxylatie tijdens bvb. het wortkoken, niet als verklarende factor. Hiervoor is een fermentatie met een Pad1-actieve giststam noodzakelijk. Deze enzym-activiteit ( phenylacrylic acid decarboxylase) werd met een grote incidentie aangetroffen in hoge gisten. De mate waarin verschillende giststammen in staat zijn om fenolzuren te decarboxyleren verschilt echter sterk. Dit maakt dat de keuze van een geschikte giststam zeer belangrijk bij de optimalisatie van vluchtige fenolen in bier. De activiteit van het Pad1-enzym is maximaal naar het einde van de gisting toe, wanneer alle vergistbare suikers bijna verbruikt zijn. Dit suggereert dat PAD1 betrokken is in het stressmetabolisme van gist dat geactiveerd wordt bij substraatuitputting. Er was een duidelijk verband tussen de fenolzuurconcentratie van wort en de uiteindelijke concentratie aan vluchtige fenolen in bier, wat er op duidt dat de optimalisatie van het fenolzuurgehalte in wort een realistische optie is wanneer men het gehalte aan vluchtige fenolen wenst te beheersen. Fermentaties op pilootschaal toonden aan dat technieken geassocieerd met modern gistmanagement in cilindroconische fermentatietanken, een grote invloed hebben op het gehalte aan 4VG in bier. Finaal werden de oorzaken van de daling van de 4VG concentratie tijdens de bierbewaring achterhaald. Een nieuwe vanille-achtige component in bier, apocynol, werd geïdentificeerd als het belangrijkste degradatieproduct van 4VG tijdens de bierveroudering. Deze component wordt gevormd door de zuur-gekatalyseerde hydratatie van de dubbele binding in de zijketen op de aromatische ring van 4VG. Wanneer er zuurstof aanwezig is in de flessenhals tijdens de bierbewaring kan 4VG ook deels geoxideerd worden tot vanilline. Beide reactiemechanismen kunnen aanleiding geven tot het vanille-achtig aroma dat ontstaat in sommige bieren tijdens de bewaring. Among the flavour-active volatile phenols in beer, most of them originate from the raw materials used in the brewing process. Only some of them can be formed by yeast activity, namely 4-vinylguaiacol (4VG) and 4-vinylphenol (4VP). The presence of these volatile phenolic compounds is considered undesirable when present in excessive concentration in bottom-fermented pilsner beers, hence the term “phenolic off-flavour” (POF). It is attributed to beers with a strong medicinal, clove-like aroma. Despite being historically catalogued as an off-flavour, these compounds are known to be essential flavour contributors to the characteristic aroma of Belgian white beers (made with unmalted wheat), German Weizen beers (made with malted wheat) and Rauch beers. In recent years, volatile phenolic flavour compounds have been increasingly encountered in Belgian specialty beers. While some brewers wish for a clear phenolic note in (some of) their beers, others do not aim for their, sometimes overwhelming, presence in the taste pallet of beer. Little is known of the dynamics behind the release of their precursors during brewing, the interactive role of native barley enzymes underlying this release and the subsequent decarboxylation to the flavour-active compounds during wort fermentation. Also the causes of the temperature dependent decrease of 4VG during beer conservation need to be elucidated. The suitability of a simple and rapid isocratic RP-HPLC method with amperometric detection for the simultaneous detection and quantification of hydroxycinnamic acids and their corresponding aroma-active volatile phenols in wort and beer is developed and validated. The method was used to perform an extensive survey on the occurrence of hydroxycinnamic acids (HCA) and volatile phenols in a range of beer styles. Odour and flavour thresholds of 4VG determined in a diverse range of beer styles confirmed the contribution of 4VG to the overall flavour perception of many top-fermented specialty beers. HCA and volatile phenols are monitored through out the beer production process to identify realistic control points for the final volatile phenol level in beer. A large variability in HCA content between different barley malt varieties and their corresponding worts was observed. Differences were also found between free FA levels from identical malt varieties originating from different malt houses. This demonstrates the importance of selecting a suitable malt variety. It was shown that only a small part of the HCA in malt is transferred to wort during mashing, while the lion share remains in the spent grains. Free HCA in wort are both water-extracted and enzymatically released by cinnamoyl esterase activity. Esterase activities clearly differ between different barley malt varieties, as do other AX-degrading enzyme activities. The release of ferulic acid during mashing did not only depend on the esterase activity, but also on the amount of esterbound ferulic acid initially present in the wort and on the endoxylanase activity. Apart from the choice of a suitable barley malt variety, final HCA concentrations in wort are also seriously affected by brew house operations. A clear difference in temperature and pH dependence between the release of the water-extracted and enzymatically hydrolysed fraction was found. In contrast to the water-extracted fraction, the hydrolysis of esterbound FA is subject to close technological control. To contribute to the odour and taste pallet of specialty beers, HCA have to be decarboxylated to the corresponding volatile phenols. Concerning thermal decarboxylation in pilsner beer, the combined time of wort boiling, transfer, whirlpool and pasteurisation times can give rise to the 4VG concentrations observed in the survey. However, the high concentrations often encountered in blond and dark specialty beers must originate from the enzymatic decarboxylation of HCA by Saccharomyces cerevisiae yeast strains during wort fermentation. The differences in volatile phenol content in top-fermented specialty beers are reflected in the high incidence of Pad1 phenotype among top-fermenting brewing yeasts strains and the observed differences in Pad1 activity between different brewing strains. Clearly, the first means for optimising the volatile phenol content in beer is the choice of a suitable yeast strains. During alcohol fermentation, it was clearly shown that the majority of 4VG is formed during the second half of the fermentation process when all fermentable sugars were consumed. This suggests that the Pad1 enzyme might be involved in the course of events triggered upon the stress presented by nutrient depletion. Clearly, elevated initial FA concentration in wort led to an increase in the 4VG content of beer. This result is highly relevant, since it validates the possibility of optimising the final volatile phenol content in beer by controlling the release of HCA in the brew house. Pilot-scale fermentation experiments showed that yeast management systems frequently encountered with cilindroconical tank fermentations sincerely affect 4VG formation during wort fermentation. Finally, the decrease in 4VG during beer conservation was examined. Two reaction mechanisms explaining the decrease of 4VG during beer ageing were identified. A new vanilla-like compound in beer, apocynol, was identified by GC-MS and HPLC-ECD analysis as the main degradation product. Apocynol is formed by an acid-catalysed hydration of the double bond of the vinyl side chain of 4VG. In the presence of oxygen, substantial amounts of vanillin were also detected. Since both apocynol and vanillin have a clear vanilla-like aroma, the decrease of 4VG during beer conservation and ageing may impart a shift from a clove-like aroma in a fresh specialty beer to a more sweet, vanilla-like flavour impression of aged specialty beer. Doctor in de Bio-ingenieurswetenschappen Centr. Levensmidd.- & Microb. Technol. Dept. Microbiële & Moleculaire Systemen Faculteit Bio-ingenieurswetenschappen Doctoral thesis Doctoraatsthesis
... Initially, a decarboxylase enzyme yields the 4-vinyl derivative. The second reaction is a reduction to form the 4ethyl derivative (Chatonnet et al. 1992;Donaghy et al. 1999). The vinylphenol reductase (VPR) enzyme putatively responsible for this step has never been isolated. ...
... The importance of this observation is that many yeast and bacteria associated with alcoholic fermentation can convert HCAs into vinyl derivatives (Degrassi et al. 1995;Rosazza et al. 1995;McMurrough et al. 1996;Donaghy et al. 1999;Karmakar et al. 2000). Accordingly, any 4-vinylphenol or 4-vinylguaiacol which has accumulated in beverages at the end of ferment through the action of other microbes could be acted on by Dekkera spoilage organisms to produce the highly undesirable 4-ethylphenol and 4-ethylguaiacol. ...
Dekkera and Brettanomyces yeast are important spoilage organisms in a number of food and beverage products. Isolates of both genera were cultured in a defined medium and supplemented with hydroxycinnamic acids and vinylphenols to investigate their influence on growth and the formation of ethyl phenol derivatives. The growth rate of Brettanomyces species in the presence of acids was reduced, and no significant conversion to vinyl or ethyl derivatives was observed. The growth rate and substrate utilisation rates of Dekkera anomala and Dekkera bruxellensis yeast differed depending on strain and the acid precursor present. Growth of D. bruxellensis was slowed by the presence of ferulic acid with the addition of 1 mM ferulic acid completely inhibiting growth. This study provides an insight into the spoilage potential of these organisms and possible control strategies involving hydroxycinnamic acids.
... An alternative catabolic route has been described for ferulic acid bioconversion in Rhodotorula rubra [17], Candida guilliermondii [18], and Cupriavidus sp. B-8 [19]. ...
Background
Lignin is an attractive alternative for producing biobased chemicals. It is the second major component of the plant cell wall and is an abundant natural source of aromatic compounds. Lignin degradation using microbial oxidative enzymes that depolymerize lignin and catabolize aromatic compounds into central metabolic intermediates is a promising strategy for lignin valorization. However, the intrinsic heterogeneity and recalcitrance of lignin severely hinder its biocatalytic conversion. In this context, examining microbial degradation systems can provide a fundamental understanding of the pathways and enzymes that are useful for lignin conversion into biotechnologically relevant compounds.
Results
Lignin-degrading catabolism of a novel Rhodosporidium fluviale strain LM-2 was characterized using multi-omic strategies. This strain was previously isolated from a ligninolytic microbial consortium and presents a set of enzymes related to lignin depolymerization and aromatic compound catabolism. Furthermore, two catabolic routes for producing 4-vinyl guaiacol and vanillin were identified in R. fluviale LM-2.
Conclusions
The multi-omic analysis of R. fluviale LM-2, the first for this species, elucidated a repertoire of genes, transcripts, and secreted proteins involved in lignin degradation. This study expands the understanding of ligninolytic metabolism in a non-conventional yeast, which has the potential for future genetic manipulation. Moreover, this work unveiled critical pathways and enzymes that can be exported to other systems, including model organisms, for lignin valorization.
... 100 From the figures in their publication, which utilise two x-axes without specification of which axis the data corresponds with, it is initially unclear as to the concentration of the glucose esters, although the initial data points correspond with those described in their previous publication, 131 Though there exists very little information on the breakdown of the glucose esters, potential pathways to degradation during the vinification process include the use of enzyme preparations containing the appropriate esterases, 64,112 or by the activity of microbiological esterases, either by those of intended wine microflora or by those of unwanted microorganisms. [135][136] Finally, the acidic environment of the wine could promote acid-catalysed hydrolysis, 135 though the results of Monagas imply that the glucose esters are stable, or that any hydrolysis is in equilibrium with re-formation. 100 To date, no research has been performed on this class of compounds with respect to the potential for breakdown by D. bruxellensis into volatile phenols. ...
The presence of esterified hydroxycinnamate conjugates in grapes and wine is well established and they account for a large proportion of the hydroxycinnamate content. There exists the possibility that these conjugates could also provide the potential for spoilage, though they have never been linked to the direct formation of ethylphenols. This research highlighted the potential role of a number of esterified conjugates in the production of ethylphenols by D. bruxellensis. Two classes of berry derived esters, the tartaric acid and glucose bound hydroxycinnamates, as well as the vinification formed ethyl esters, were synthesised and used for model fermentation experiments.
... Des activités AFE ont également été observées chez certaines levures telles que Candida lambica, Rhodotorula rubra/glutinis, Rhodotorula minuta (Donaghy et al., 1999) et récemment chez Saccharomyces cerevisiae (Coghe et al., 2004). De plus, certaines plantes possèdent des activités AFE, notamment l'orge (Sancho et al., 1999 ;Sancho et al., 2001). ...
L'induction de la synthèse d'une acide férulique estérase (AFE) a été étudiée chez Streptomyces ambofaciens ATCC 23877. L'activité la plus élevée a été détectée en présence de son de blé désamidonné ou de xylane d'avoine (0,22, 0,21 mU/mg protéine, respectivement). Des productions d'AFE en bioréacteur ont également été réalisées en utilisant 1% (p/v) de son de blé comme inducteur. Le niveau de production de l'AFE a été trois fois plus important en bioréacteur qu'en fiole d'Erlenmeyer. L'AFE de Streptomyces ambofaciens ATCC 23877 et celle de Humicola sp., présente dans un mélange enzymatique commercial (DepolTM 740L), ont été partiellement purifiées et caractérisées. A l'issue de la purification, l'activité AFE de Streptomyces ambofaciens ATCC 23877 a été trop faible pour pouvoir être utilisée ultérieurement en synthèse. Par contre, le potentiel de l'AFE de Humicola sp., concentrée par précipitation à l'acétone, pour la synthèse de différents esters phénoliques a été testé. Les meilleurs rendements de conversion ont été observés lors de l'absence de substitutions sur le cycle aromatique de l'acide phénolique ou en présence de groupements hydroxyles. Les synthèses en milieu non aqueux (M2B2) se sont montrées infructueuses en raison, peut-être, d'un effet néfaste du solvant sur l'enzyme
... Rhodotorula rubra), Rhodotorula minuta and Pichia fermentans (syn. Candida lambica) [103] were able to transform ferulic acid into 4-vinylguaiacol. ...
... This compound can be obtained by microwave-assisted extraction, an ecofriendly chemical synthesis (Bernini et al. 2007), although microbial decarboxylation of cinnamic acids is preferred, in that it is a cheaper and cleaner process. Although some yeasts (Donaghy et al. 1999;Mathew et al. 2007) and fungi (Topakas et al. 2003) have been evaluated here, bacteria are the microorganisms most widely assayed in this way. Thus several species, such as S. setonii Salgado et al. 2012), A. niger (Baqueiro-Peña et al. 2010), Enterobacter sp. ...
A strain designated as BA03, with the ability to transform ferulic acid into vanillin and 4-vinylguaiacol, was isolated from contaminated cryovials. The production of natural value-added compounds was dependent on the media employed. The morphological and physiological characteristics of this strain were compared with those of the typical vanillin-producer strain Amycolatopsis sp. ATCC 39116. According to a partial 16S rRNA sequence, we determined that BA03 belonged to Bacillus aryabhattai. In addition, analysis of the results showed that this strain exhibited interesting enzymatic activity, including cellulases, laccases, lipases and pectinases. In light of this, we propose new functions for this multitasking microorganism. We suggest that it may be used for converting lignocellulosic wastes into byproducts with industrial uses, and also for treating disposal residues such as dyes in the textile industry. Hence, the possibility for novel research with B. aryabhattai opens up in the fields of biodegradation and/or revalorization of wastes.
... One gram quantity of uninoculated and A. niger grown wheat and rice bran samples of varying incubation periods were extracted with extraction medium (6.6 ml), containing Tris (hydroxymethyl) aminomethane hydrochloride, 50 mM, pH 8.0, reduced glutathione, 25mM, Triton-X-100, 1% (wt/vol), polyvinylpolypyrrolidone, 0.2 g for 2 h at 4 0 C, centrifuged, dialyzed against tris buffer (50 mM, 8.0 pH). Ferulic acid esterase activities were assayed using ethyl ferulate as the substrate, incubating at 37 0 C for 1 h (Donaghy et al., 1999). ...
... Rhodotorula rubra), Rhodotorula minuta and Pichia fermentans (syn. Candida lambica) [103] were able to transform ferulic acid into 4-vinylguaiacol. ...
The rising consumer requests for natural flavors and fragrances have generated great interest in the aroma industry to seek new methods to obtain fragrance and flavor compounds naturally. An alternative and attractive route for these compounds is based on bio-transformations. In this review, the application of biocatalysis by Non Conventional Yeasts (NCYs) whole cells for the production of flavor and fragrances is illustrated by a discussion of the production of different class of compounds, namely Aldehydes, Ketones and related compounds, Alcohols, Lactones, Terpenes and Terpenoids, Alkenes, and Phenols.
... besides its commercial value as a flavor and fragrance substance, 4-VG has medicinal value due to its antioxidant activity (bortolomeazzi et al. 2007; terpinc et al. 2011) and possible anti-cancer activity (Jeong & Jeong 2010). a wide variety of aromatic compounds can be produced using ferulic acid metabolism by four major pathways, namely i) non-oxidative decarboxylation, ii) side chain reduction, iii) coenzyme-a-dependent deacetylation, and iV) coenzyme-a-independent deacetylation. of these, 4-VG is obtained by nonoxidative decarboxylation of ferulic acid (rosazza et al. 1995;Lee et al. 1998;donaghy et al. 1999). the bioconversion of ferulic acid to 4-VG has been studied using a variety of microorganisms, such as Bacillus coagulans, Debaryomyces hansenii, white rot fungus, Aspergillus niger, Lactobacillus farciminis, Streptomyces setonii, Enterobacter soli, and E. aerogenes (karmakar et al. 2000;Mathew et al. 2007;Mabinya et al. 2010;baqueiro-Peña et al. 2010;adamu et al. 2012;Max et al. 2012;hunter et al. 2012). ...
Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.
... 33 Also, yeasts have the ability to produce ferulic acid decarboxylase and metabolise ferulic acid to 4-vinyl guaiacol. 34 Such transformation of free PAs could take place during fermentation of doughs E as well, since their amounts found in these breads (33-45 and 11-15 μg g −1 DM) were lower or comparable to those of breads D. Rye wholemeal breads contained 12-52 μg g −1 DM free ferulic acid, 4-16 μg g −1 DM sinapic acid and 2-8 μg g −1 DM vanillic acid (Fig. 2). The remaining PAs identified (caffeic, p-coumaric and p-hydroxybenzoic acids) and vanillin were the minor components. ...
Background
Bread-making process influences bread components, including phenolics that significantly contribute to its antioxidant properties. Five bread model systems made from different rye cultivars were investigated to compare their impact on concentration of ethanol-soluble (free and ester-bound) and insoluble phenolics.ResultsBreads produced by a straight dough method without acid addition (A) and three-stage sourdough method with 12-h native starter preparation (C) exhibited the highest, genotype-dependent concentrations of free phenolic acids. Dough acidification by direct acid addition (method B) or by gradual production during prolonged starter fermentation (24 and 48 h, for methods D and E) considerably decreased their level. However, breads B were enriched in soluble ester-bound fraction. Both direct methods, despite substantial differences in dough pH, caused a similar increase in the amount of insoluble ester-bound fraction. The contents of phenolic fractions in rye bread were positively related to activity level of feruloyl esterase and negatively to those of arabinoxylan-hydrolyzing enzymes in whole-meal flour.Conclusion
The solubility of rye bread phenolics may be enhanced by application of a suitable bread-making procedure with respect to rye cultivar, as the mechanisms of this process are also governed by a response of an individual genotype with specific biochemical profile.
... Bacillus coagulans BK07 Karmakar et al. (2000) 4-vinyl guaiacol Rhodotorula rubra, Rhodotorula minuta and Candida lambica Donaghy et al. (1999) 4-vinyl guaiacol Pycnoporus cinnabarinus MUCL39533 Bonnin et al. (1999) 4-vinyl guaiacol Bacillus pumilus DRV 52131 (NRRL B-14942) Lee et al. (1998) 4-vinyl guaiacol Bacillus pumilus Degrassi et al. (1995) 4-vinyl guaiacol Pseudomonas fluorescens Huang et al. (1994) ...
The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.
... These results indicated that wheat Qu indeed plays an important role in the production of FA and 4-VG in Chinese rice wine brewing. Some yeast strains have been reported to produce both a feruloyl esterase for the release of FA and a FA decarboxylase for the formation of 4-VG 10 . However, feruloyl esterase activity in S. cerevisiae has not been previously reported and it was not present in the Chinese rice wine yeast in this study (data not shown). ...
The release of ferulic acid (FA) and the subsequent enzymatic decarboxylation to 4-vinylguaiacol (4-VG) were nearly ubiquitous during the production of Chinese rice wine. To evaluate the release of FA and the transformation to 4-VG, the levels of FA and 4-VG were determined by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrum (GC-MS), respectively. During rice wine brewing, the concentrations of FA and 4-VG increased as fermentation time prolonged, with a rapid rise in days 1 to 5 of the main fermentation and followed by a slower rise from the 6th day post inoculation. FA and 4-VG levels in mashes, fermented by yeast with wheat Qu, were significantly higher than those in mashes fermented by yeast using commercial enzyme additions. The release of FA from rice by wheat Qu involves an enzymatic release mechanism. The levels of FA and 4-VG from rice flour by wheat Qu rose gradually during the first 24 h and then tended to stabilize. The capacity of yeasts to decarboxylate phenolic acids (Pof+ phenotype) was absent in most rice wine brewing yeasts. These results suggest that the production of FA and 4-VG originated from materials in the wheat Qu rather than by the yeast during Chinese rice wine brewing and fermentation.
... One gram quantity of uninoculated and A. niger grown wheat and rice bran samples of varying incubation periods were extracted with extraction medium (6.6 ml); containing tris (hydroxymethyl) aminomethane hydrochloride, 50 mM, pH 8.0; reduced glutathione, 25 mM; Triton-X-100, 1% (wtnvol); polyvinylpolypyrrolidone, 0.2 g for 2 h at 4°C, centrifuged, dialyzed against tris buffer (50 mM, 8.0 pH). Ferulic acid esterase activities were assayed using ethyl ferulate as the substrate, incubating at 37°C for 1 h (Donaghy, Kelly, & McKay, 1999). Reaction was stopped by the addition of methanol. ...
Cereal brans such as wheat and rice are abundant sources for obtaining bioactive phenolic compounds such as ferulic and cou-maric acids, in turn these can be bio-transformed into high value flavour compounds such as vanillin. Aspergillus niger CFR 1105, found to induce greater amounts of cell wall degrading enzymes, was inoculated into wheat and rice brans, and grown for 24, 48, 72 and 96 h to understand the degradation pattern of non-starch polysaccharides and phenolic acid complexes. Native wheat bran polysaccharides mainly consisted of arabinose, xylose, galactose, and glucose, in % of 27:39:2:30, with traces of mannose, whereas rice bran consisted of arabinose, xylose, galactose, and glucose in % of 9:27:31:32, respectively. Both the cereal bran arabinoxylans were degraded extensively by A. niger at 96 h, whereas the degradation of 1,3/1,4-b-D-glucans and cellulose was negligible. Bound phenolic acids of rice and wheat brans identified by HPLC were found to be mainly ferulic, coumaric, syringic acids in % of 93.6:6.3:0 (wheat), 34.7:55.8:9.5 (rice) and were drastically degraded/utilized at 96 h. The above results have indicated preferential degradation of arabinoxylans of wheat bran by A. niger, which can be exploited to obtain bioactive compounds such as ferulic acid.
... Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is an abundant, ubiquitous cinnamic acid found throughout the plant kingdom (Andreoni et al. 1995;Bouaziz and Sayadi 2005;Donaghy et al. 1999). There is a growing interest in the potential use of ferulic acid for biocatalytic conversion into other high-added-value products such as vanillin, vanillic acid, 4-vinylguaiacol and styrenes (Bonnin et al. 2001;Ghosh et al. 2007). ...
During growth on ferulic acid, Halomonas elongata DSM 2581T was capable of promoting the formation of a significant amount of vanillic acid. The products were confirmed by high-performance
liquid chromatography and gas chromatography mass-spectrometry analyses. To enhance the formation of vanillic acid and prevent
its degradation, a resting-cell method using Halomonas elongata was developed. The growth state of the culture utilized for biomass production, the concentration of the biomass, the amount
of ferulic acid that was treated and the reutilization of the biomass were optimized. The optimal yield of vanillic acid (82%)
was obtained after a 10-h reaction using 10 mM ferulic acid and 5 g/l of cell pregrown on ferulic acid and harvested at the
end of the exponential phase.
... The thermal and enzymatic decarboxylation of 4-hydroxycinnamic acids [4-hydroxycinnamic (p-coumaric); 3,4-dihydroxycinnamic (caffeic); 3-methoxy- 4-hydroxycinnamic (ferulic), and 3,5-dimethoxy-4-hydroxycinnamic (sinapic) acids] results in the formation of the respective 4-hydroxystyrenes [4-vinylphenol, 3,4-dihydroxystyrene, 3-methoxy-4-vinylphenol (4-vinylguaiacol), and 2,6-dime- thoxyvinylphenol (4-vinyl syringol)]. These decarboxylation products and their adducts are present in a diverse range of beverages and foods, including various wines, beers, juices, and berries (Pyysalo et al., 1977; Heresztyn, 1986; Donaghy et al., 1999; Suarez et al., 2007; Vanbeneden et al., 2008; Callemein & Collin, 2010). The thermal decarboxylation of 4-hydroxycinnamic acids has been studied since the 1940s with various catalysts, including copper/quinoline (Walling & Wolfstirn, 1947), KCl (Cohen & Jones, 1960), amines (Nomura et al., 2005), and sodium acetate (Terpinc et al., 2011). ...
This chapter highlights that the ability to protect oil-in-water emulsion systems from oxidative deterioration has growing importance for the food and nutraceutical industries as new products are entering the market that have both enhanced levels of unsaturated Fatty Acids (FAs) and reduced levels of saturated and trans fats. Canola (Brassica napus) is the major oilseed crop grown within Australia and naturally contains high levels of antioxidants, including both α- and γ-tocopherols and phenolic acids. The predominant phenolic acid found in all Brassica oilseeds is sinapic acid and the major mechanism for the antioxidant activity of phenolic acids derives from their ability to scavenge free radicals by donating their phenolic hydrogen atoms. The resulting phenolic radical is resonance stabilized, with various functional groups either increasing or reducing this resonance stabilization. Tocopherols use a similar mechanism to protect against oxidative deterioration. Preheating canola seeds prior to oil extraction can further improve their inherent oxidative stability within bulk-oil applications. This is partly because the thermal decarboxylation of sinapic acid forms a potent lipophilic antioxidant, 2,6-dimethoxyvinylphenol, also known as 4-vinyl syringol (4-VS) or canolol. The chapter provides the details pertaining to this mechanism. Further, results from the study reported in the chapter show that fortification can improve the oxidative stability of 10% water-in-water tuna oil-in-water emulsions systems at levels of 5, 10, and 20% water-in-water total oil phase. Moreover, the results indicate that oil from preheated seed can provide protection from oxidative deterioration that is as good as or, in some cases, better than a selection of commonly used antioxidants. © 2013 AOCS Press Published by Elsevier Inc. All rights reserved.
... i) Nonoxidative decarboxylation to give 4-vinylguaiacol (4- hydroxy-3-methoxystyrene) (Rosazza et al., 1995; Lee et al., 1998; Donaghy et al., 1999). *Corresponding author. ...
A white rot fungus isolated from decaying wood was investigated for its ability to convert ferulic acid to various valuable products. The fungus is able to convert ferulic acid to 4-vinylguaiacol, which is then metabolized further to acetovanillone. Both products have potential use in the chemical manufacturing and pharmaceutical industries and these results contribute to our knowledge of the biotransformation of ferulic acid.
... The optimal temperature and pH of PAD activity for D. bruxellensis was investigated for the first time in the present work and found to be almost identical to that of D. anomala at pH 6 and 40°C. These parameters are similar to those determined for Saccharomyces and Candida yeast (Donaghy et al. 1999;Goodey and Tubb 1982). ...
Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.
... Ferulic acid is also a precursor for 4-vinyl guaiacol (3methoxy 4-hydroxystyrene), the transformation being catalyzed by the enzyme ferulic acid decarboxylase (Donaghy et al. 1999). 4-vinyl guaicol is nearly 25-30 times more costlier than ferulic acid which makes the bioconversion, a highly value added process. ...
Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and is ester linked to arabinose, in various plant polysaccharides such as arabinoxylans and pectins. It is a precursor to vanillin, one of the most important aromatic flavor compound used in foods, beverages, pharmaceuticals, and perfumes. This article presents an overview of the various biocatalytic routes, focusing on the relevant biotransformations of ferulic acid using plant sources, microorganisms, and enzymes.
... strain B1 (Peng et al., 2003). Many works showed the degradation of ferulic acid by one-carbon cleavage of the side chain to form 4-vinylguaiacol (Edlin et al., 1995;Donaghy et al., 1999;Karmakar et al., 2000). ...
Halomonas elongata strain Mar (=CCUG 52759) isolated from table-olive fermentation is the first halophilic bacterium to be shown to transform ferulic acid to vanillic acid under hypersaline conditions. During growth on ferulic acid, this strain was capable of promoting the formation of a significant amount of vanillic acid and trace quantities of vanillin. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Based on the different metabolites identified, an oxidative side chain degradation pathway of ferulic acid bioconversion to vanillic acid was suggested. Phylogenetic analysis of 16S rRNA gene revealed that this isolated strain Mar was identified as H. elongata. To increase the formation of vanillic acid, a resting cell method using H. elongata strain Mar was performed. The optimal yield of vanillic acid (86%) was obtained after a 6 h reaction using 5 mM of ferulic acid and 4 g of dry weight of cells L(-1) pregrown on ferulic acid and harvested at the end of the exponential phase.
The development of biorenewable resources to substitute for oil-based feedstocks in polymer manufacture is essential to address sustainability. Ethylene, propylene, and styrene together represent ~90% of the feedstocks used by the polymer industry. Some industries have attempted to utilize bio-based ethylene and propylene. However, there are very few known biosynthetic pathways to produce styrene and, generally, they are not well understood. The decarboxylation of cinnamic acid by ferulic acid decarboxylase (FDC) is one of the few biological pathways known that form styrene. The focus of this dissertation is to explore the recently proposed FDC decarboxylation mechanisms and cofactor specificity of the enzyme. The initially proposed mechanisms for FDC decarboxylation of phenylacrylic acid postulated either Michael addition or 1,3-dipolar cycloaddition. 1H NMR analysis, isotope effects and linear free-energy analysis were employed to further investigate the proposed mechanisms and determine the rate-determining step in the reaction. 1H NMR experiments demonstrated that FDC decarboxylation was stereospecific and the source of proton that replaced the carboxylic group of phenylacrylic acid was the solvent. Proton inventory experiments suggested that a single proton was involved in the transition state. The negative Hammett reaction constant provided evidence that the 1,3-cycloelimination step in the 1,3-dipolar cycloaddition mechanism is likely to be rate-determining in the FDC decarboxylation. This was further supported by secondary kinetic isotope effect experiments. xv To investigate the biosynthesis of the prFMN cofactor by prenyl flavin synthase (PFS), a scPFSFDC coupled assay was developed and optimized to monitor scPFS prenylation through the activation of FDC. With scPFS-FDC coupling assay, we found that scPFS selectively uses dimethylallyl pyrophosphate as the substrate for FMN prenylation, in contrast to the bacterial enzyme for which dimethylallyl monophosphate was reported to be the substrate. By implementing this coupled assay, steady-state kinetic parameters of scPFS prenylation were obtained. Commercially available substrate analogs, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and geranyl pyrophosphate (GPP), were used to investigate the substrate specificity of scPFS prenylation. The apparent first-order rate constants of prenylation were 4.45 ± 0.87 h-1, and 3.80 ± 0.34 h-1 when HMBPP and GPP were respectively used as substrate under single turnover kinetics. Under steady state conditions, FDC was shown to utilize the cofactor synthesized from HMBPP to decarboxylate cinnamic acid at Vmax = 5.50 ± 0.37 min-1. However, in the case of GPP counterpart, no decarboxylation activity was observed. The results presented in this thesis provide better understanding of styrene biosynthesis by FDC. In order to be implemented in bioindustry, this system would have to be further optimized, especially in terms of enzymatic efficiency. One proposed strategy is the use of cofactor analogs. Preliminary investigations here have enabled to establish the procedure that would ultimately lead to the discovery of cofactor analogs with enhanced decarboxylation activity.
A decarboxylase (IfPAD) from the ascomycete Isaria farinosa converted ferulic acid to 4-vinylguaiacol (4-VG), a volatile which imparts the distinct “smoke flavor” of pyrolized wood. The activity was enhanced by adding (E)-ferulic acid to the culture medium and peaked with 3.6 U g⁻¹ mycelium (one μmol 4-VG min⁻¹) after five days of submerged cultivation. The coding sequence of 543 bp was translated into a 25 kDa protein with 180 aa and a homology of 91 % to putative phenolic acid decarboxylases of its teleomorph, Cordyceps militaris, and Beauveria bassiana, the anamorph of Cordyceps bassiana. Cold shock expression in E. coli yielded an activity of 411 U g⁻¹ wet mass. Substrate conversion required a hydroxyl substituent para to a trans-unsaturated C3-side chain of the aromatic ring. Km and kcat/Km values were determined to 0.3 mM and 78.4 mM⁻¹s⁻¹ for p-coumaric acid and 1.9 mM and 45.1 mM⁻¹s⁻¹ for (E)-ferulic acid, respectively. The native enzyme and its recombinant counterpart showed pH-optima at pH 6.0 and pH 5.5, and low temperature optima of 19 °C and 14 °C, respectively. IfPAD produced 4-VG from destarched wheat bran and sugar beet fiber, confirming activity on complex plant biomass. The enzyme opens a route to the cold generation of smoke flavor without the risk of formation of polycyclic aromatic hydrocarbons or other adverse side-products, which inevitably occur during the traditional pyrolysis of wood. This is the first report on the biochemical characterization of a phenolic acid decarboxylase from a filamentous ascomycete.
Fonseca, B.G. Evaluation of biological treatment conditions of hemicellulosic hydrolysates aiming to improve ethanol production by Scheffersomyces (Pichia) stipitis 2014. 186p. Thesis (Doctoral of Science) – Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, São Paulo.
The aim of this work was to study the ability of Saccharomyces cerevisiae to metabolize a variety of toxic compounds found in rice straw (RSHH) and olive tree pruning (OTHH) hemicellulosic hydrolysates, in order to obtain hydrolysates with lower toxicity for Pichia stipitis. After determined the hydrolysate composition (sugar and toxic compounds) was evaluated the RSHH concentration level able to inhibit the P. stipitis metabolism. Then, the effect of time, S. cerevisiae concentration, pH and aeration on the hydrolysates composition was evaluated, and the fermentation was used as response. Under optimized conditions of biotreatment, P. stipitis fermentations were conducted in shake flasks and in bioreactor. In relation to the biotransformation assays of these hydrolysates, as well as synthetic medium (SM), with the same sugar and toxic compounds concentrations found in RSHH, the results showed that changes in the media compositions were dependent on the treatment time. During the treatment, S. cerevisiae consumed only D-glucose with low ethanol, glycerol, and acetic acid production. Furthermore, in SM and RSHH media, 5-HMF and furfural were almost completely assimilated (> 90 %), with low levels of furoic acid formation. In these media, S. cerevisiae partially converted ferulic acid (15%) and p-coumaric acid (20%), being observed only the conversion product of ferulic acid (vanillyl alcohol). Vanillin was totally assimilated in SM, however a residual of this compound (42%) was observed in RSHH, being vanillyl alcohol the main conversion product. Regarding OTHH was observed the assimilation of total furans (47%) and total phenolic (11%), and no conversion products were identified. The results from the P. stipitis fermentation in RSHH showed that biotreatment for 6 hours favored D-xylose consumption and ethanol production, indicating that this parameter is an important factor to be considered. In these conditions, D-xylose consumption was 57% with 9 g/L ethanol production (YP/S = 0.18 g/g QP = 0.086 g/Lh). The results of OTHH fermentation showed lower YP/S (0.14 g/g) and QP (0.060 g/L.h ) compared to RSHH, which indicates a higher degree of toxicity in OTHH. Regarding SM, the biotreatment increased D-xylose consumption and ethanol production by P. stipitis in approximately 40%, when compared with untreated-SM. The most suitable conditions of S. cerevisiae concentration (5 g/ L), pH (3.0) and the aeration factor (6.5) defined in RSHH through an experimental design, provided 67% of D-xylose consumption, and 13.5 g/L ethanol production by P. stipitis (YP/S = 0.24 g/g QP = 0.15 g/L.h). In bioreactor, P. stipitis was able to completely consume D-xylose, producing 23 g/L ethanol after 44 hours. Based on these results, it can be concluded that previous treatment of hemicellulose hydrolysates with S. cerevisiae is a promising technique capable of reducing the toxicity degree of RSHH and OTHH, with consequent improvement in their fermentability, especially on ethanol production rate.
Keywords: Ethanol, Scheffersomyces (Pichia) stipitis, hemicellulosic hydrolysate, biotreatment, Saccharomyces cerevisiae.
This paper summarises the occurrence in foods and beverages of the cinnamic acids, their associated conjugates and transformation products. Quantitative data are lacking for some commodities known to contain them, but it is clear that for many people coffee will be the major source. The daily dietary intake of total cinnamates may vary substantially from almost zero to perhaps close to 1 g. The data relating to their absorption and metabolism are presented along with a consideration of their possible in vivo effects. Data for true bioavailability are incomplete: in particular it is not clear whether availability differs markedly with the form of the conjugate, and whether as a consequence some dietary sources may be superior to others. (C) 2000 Society of Chemical Industry.
The microbial transformation of ferulic acid (FA) offers a cleaner, more economical alternative for the natural production of flavorings and fragrances. In the present study, the biotransformation of FA using the filamentous phytopathogenic fungi Colletotrichum acutatum and Lasiodiplodia theobromae was researched. Initially, the toxicity of FA against both fungi was evaluated; the FA displayed a moderate toxicity (total inhibition at concentrations ≥ 2000 mg L-1) and apparently a detoxification mechanism was present. Afterwards, the microorganisms were incubated with the substrate at room conditions using a Czapek-Dox culture medium. The results demonstrated that the FA was mainly converted to 4-vinylguaiacol, reaching the highest abundance within the first 48 hours. To a lesser extent, acetovanillone, ethylguaiacol, and vanillin, among others, were produced. Interestingly, the compounds generated in the biotransformation of FA with C. acutatum and L. theobromae have been used as flavorings. Based on the identified metabolites, a possible metabolic pathway was proposed.
The trend toward natural flavors has led to extensive research in this area. In the biotechnological process, ferulic acid was commonly used as a substrate and employs microorganisms as a biocatalyst. In this study, the potential of three white rot fungi- Ganoderma mastoporum, Ganoderma australe and Trametes pavonia- isolated from the fruiting-body of the polypore mushroom and two strains of commercial mushrooms (Ganoderma lucidum and Schizophyllum commune) were investigated for their ability to convert ferulic acid into its metabolites using GC-MS. The results showed that G. lucidum, S. commune and T. pavonia were able to convert ferulic acid to 4-vinyl guaiacol as a major degradation product, while the extract of G. australe and G. mastoporum grown in the same medium were found to obtain vanillin as a major component. Moreover, various degradation products, including methoxy benzoquinone, 2-methoxyhydroquinone and methyl eugenol, were also found in this experiment.
Volatile phenols have been considered as a class of highly flavour-active compounds in non-alcoholic and alcoholic beverages. However, the information on volatile phenols in Chinese liquors remains limited. This article presents a high-performance liquid chromatography with a fluorescence detector (HPLC-FLD) method for the determination of 10 volatile phenols in Chinese liquors. The method was based on the fluorescent nature of volatile phenols, the separation of the isomers p-cresol and m-cresol with β-cyclodextrin as the resolving agent, and the use of a protective barrier layer for the capture of volatile phenols during the concentration process of the extract solution. The effects of several experimental parameters on recoveries of target volatile phenols were investigated. The correlation coefficients (R2) for calibration curves of the 10 phenols studies were in the range 0.9991–0.9998 when the linearity range was from 0.05 to 4 µg/ml. The proposed RP-HPLC method was successfully applied to the analysis of Chinese liquors and the recoveries of the phenols were in the range 91.5–100.4% with RSD 0.9–2.2%, and the limits of detection were 0.0072–0.0234 µg/ml. Volatile phenols of different types and in differing amounts were found in several Chinese liquors. The method could be of use for routine evaluation of the quantity of volatile phenols during the production and ageing process of Chinese liquors. Copyright © 2013 John Wiley & Sons, Ltd.
Polyphenols and organic acids were the important constitutes in the cider because they greatly contribute to organoleptic quality. The determination of their changes is important for monitoring the fermentation process for purposes of quality control. In this study, evolution of polyphenols and organic acids was monitored throughout the cider fermentation process. The samples were taken periodically and the polyphenols and organic acids contents were determined using HPLC methods.
The contents of polyphenols and organic acids were in constant change. After fermentation, the content of (+)-catechin, (-)-epicatechin, chlorogenic acid, cinnamic acid, p-coumaric acid, gallic acid, caffeic acid, ferulic acid, rutin and phloridzin decreased by different degrees, while protocatechuic acid increased after fermentation. The content of organic acids was also affected by fermentation. Malic acid, lactic acid, quinic acid, pyruvic acid and citric acid showed different levels of increase, but succinic acid content decreased.
The content of polyphenols and organic acids were affected by fermentation. The changing profiles of them during fermentation process were dependent on the type of phenolic compounds and organic acids studied.
This paper summarises the occurrence in foods and beverages of the cinnamic acids, their associated conjugates and transformation products. Quantitative data are lacking for some commodities known to contain them, but it is clear that for many people coffee will be the major source. The daily dietary intake of total cinnamates may vary substantially from almost zero to perhaps close to 1 g. The data relating to their absorption and metabolism are presented along with a consideration of their possible in vivo effects. Data for true bioavailability are incomplete: in particular it is not clear whether availability differs markedly with the form of the conjugate, and whether as a consequence some dietary sources may be superior to others.© 2000 Society of Chemical Industry
This work deals with the screening of seven lignocellulosic residues: chestnut and pistachio shells, grass, leaf fruit, vine leaf, and, red and white grape stems as feedstocks for the release of ferulic acid by sequential pre-treatment with dilute sulfuric acid and alkaline hydrolysis with NaOH. Grass showed the highest concentration of both ferulic acid (312.0 mg/L) and p-coumaric acid (542.7 mg/L). Ferulic acid was further purified from the brown liquor by ethanol extraction, or adsorption with activated charcoal or polyvinylpolypyrrolidone (PVPP). Finally, streams coming from these treatments were fermented by Streptomyces setonii after nutrients supplementation in solid state fermentation. Ferulic acid treated with PVPP presented the best results, achieving 92.1 mg/L 4-vinyl guaiacol (3-methoxy 4-hydroxystyrene) after 30 h of fermentation (QP = 3.071 mg/L h; YP/S = 0.44 g/g), thus confirming the efficacy of the materials as renewable feedstocks, and the treatments for producing natural food additives with high add value.
Debaryomyces hansenii NRRL Y-7426 metabolised ferulic acid into different phenolic compounds using a factorial design where glucose concentration (in the range of 1–20 g/L), peptone concentration (2–20 g/L) and yeast extract concentration (0.2–10 g/L) were the independent variables. The interrelationship between dependent and operational variables was well fitted (R
2 > 0.95) to models including linear, interaction and quadratic terms. Depending on the glucose and nitrogen concentrations, which redirected the metabolism, the major degradation products were 1,226.2 mg 4-vinyl guaiacol/L after 72 h (molar yield of 86.0 %), 1,077.8 mg vanillic acid/L after 360 h (molar yield of 91.1 %) or 1,682.6 mg acetovanillone/L after 408 h (molar yield of 98.8 %) in fermentations carried out with 2,000 mg ferulic acid/L. Other metabolites such as vanillin, vanillyl alcohol or 4-ethylguaiacol were present in lower amounts.
A heat-labile phenolic acid decarboxylase from
Candida guilliermondii (an anamorph of Pichia guilliermondii)
was purified to homogeneity by simple successive
column chromatography within 3 days. The molecular
mass was 20 kDa by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis and 36 kDa by gel-filtration
chromatography, suggesting that the purified enzyme is a
homodimer. The optimal pH and temperature were
approximately 6.0 and 25�C. Characteristically, more than
50% of the optimal activity was observed at 0�C, suggesting
that this enzyme is cold-adapted. The enzyme
converted p-coumaric acid, ferulic acid, and caffeic acid to
corresponding products with high specific activities of
approximately 600, 530, and 46 U/mg, respectively. The
activity was stimulated by Mg2? ions, whereas it was
completely inhibited by Fe2?, Ni2
?, Cu2?, Hg2?, 4-chloromericuribenzoate,
N-bromosuccinimide, and diethyl
pyrocarbonate. The enzyme was inducible and expressed
inside the cells moderately by ferulic acid and p-coumaric
acid and significantly by non-metabolizable 6-hydroxy-2-
naphthoic acid.
4-Hydroxycinnamic, 4-methoxycinnamic, ferulic and cinnamic acids were both non-oxidatively and oxidatively decarboxylated in alkaline aqueous media in the presence of l-cysteine–Fe(II) and l-cysteine–Co(II) heterogeneous catalysts using hydrogen peroxide or molecular oxygen. GC/MS analysis of diethylether extracts of reaction mixtures confirmed that the addition of hydrogen peroxide resulted predominantly in oxidative decarboxylation of substituted cinnamic acids, producing the corresponding carbonyl compounds (4-hydroxybenzaldehyde, 4-methoxybenzaldehyde, vanillin, benzaldehyde). On the other hand, saturation of this heterogeneous reaction system with molecular oxygen led to the formation of a variety of products, probably via peroxoacid anions or peroxoradical intermediates, e.g., ferulic acid was transformed to vinylguaiacol and vanillin with yields of 22% and 0.7%, respectively.
Debaryomyces hansenii, an isolated yeast strain metabolized ferulic acid to an intermediate compound, namely 4-vinyl guaiacol, by the non oxidative decarboxylation of its side chain. This bioconversion is a highly value added process as 4-vinyl guaiacol is nearly 40 times costlier than ferulic acid. The major degradation products were isolated and identified by thin layer chromatography, high performance liquid chromatography and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Debaryomyces produced 1470 mg l−1 of vinyl guaiacol at the tenth hour itself, corresponding to a molar yield of 95% while the production of vanillin reached a maximum of 169 mg l−1 at the fifth hour.
This work represents a survey of the occurrence of hydroxycinnamic acids and volatile phenols in a variety of beer styles. The contribution of 4-vinylguaiacol to the overall flavour perception of top-fermented specialty beers was shown. Significant differences in hydroxycinnamic acids (both free and ester-bound) and volatile phenol content between different beers were observed. The variability in volatile phenol content between different beers and beer styles can be explained by the high incidence of Pad1+ phenotype and the variability of Pad1 activity observed among top-fermenting brewing yeast strains. The relative importance of thermal versus enzymatic decarboxylation can account for the differences found between bottom and top-fermented beers. Concerning the optimisation of volatile phenol levels in beer, the selection of a suitable brewing yeast strain is the most important means of creating a phenolic taste profile in beer. Given that a considerable amount of hydroxycinnamic acids in beer still occurs in ester-bound form, enhancing the enzymatic release of these phenolic flavour precursors during mashing can greatly enhance the phenolic aroma potential of wort.
4-vinyl guaiacol (3-methoxy 4-hydroxystyrene) can be obtained by decarboxylation of ferulic acid by the strain Streptomyces setonii ATCC 39116. The formation of this metabolite was favoured by microaerobic conditions and the culture medium employed, increasing progressively the product concentration from 543.3 up to 885.1 mg/l when aeration level was diminished, reaching a highest volumetric productivity of 70.4 mg/l h and a product yield of 1.11 mol/mol. The identity of the metabolite was confirmed by gas chromatography-mass spectrometry. A metabolic study of ferulic acid and the main degradation products (ferulic acid, 4-vinyl guaiacol, protocatechuic acid, vanillyl alcohol, vanillic acid and vanillin) suggested that ferulic acid was the only substrate capable to be transformed into 4-vinyl guaiacol by this strain of S. setonii.
Until recently, acidic products such as fruit juice and fruit based products were generally thought to be susceptible to spoilage by yeasts, mycelia fungi and lactic acid bacteria, as the low pH of these products acts as natural control measures against spoilage by most bacteria. Alicyclobacillus seem to be prevalent in fruit based products as they survive the acidic fruit juice environment, even when they are exposed to pasteurisation temperatures during production. In this review the historical background of the discovery of these bacteria is summarised. The bacterial characteristics and the reported spoilage incidences caused by members of this genus are discussed. As the isolation methods for these bacteria are controversial, this review includes a discussion of the various media that have been reported in the literature for the use in the isolation and enumeration of members of the genus Alicyclobacillus.
Volatile phenols are spoilage compounds of many foods, which have also been detected in the volatile fraction of defective virgin olive oils (VOOs). However, their formation in olive fruits or VOOs, as well as the factors affecting their production, has yet to be elucidated. In the present study, the evolution of volatile phenols was monitored for the first time in VOOs obtained from olives stored during different periods under two different conditions of limited aerobiosis. Moreover, their odor activity values (OAVs) in VOO samples were calculated as a first assessment of their sensory importance, and the microbiological profile of the olives' surface was evaluated at each sampling point in order to clarify the possible causes of volatile phenols formation. Although volatile phenols seem to acquire their sensory significance at advanced stages of olives' alteration, they were significantly correlated to the time of olives' storage and in accordance with sensory evaluation, indicating that they could be considered as analytical indices of olive fruits' degradation during storage, likely reflecting the microbiological activity.
In this study the decrease of 4-vinylguaiacol (4VG) during beer aging was investigated and the products that arise from it were identified. Two compounds, vanillin and apocynol, were identified in beer model solutions after forced aging and in naturally aged beers by GC-MS and HPLC-ECD analyses. Both account for up to 85% of the decrease of 4VG. Only in the presence of substantial amounts of oxygen in the bottle headspace was vanillin detected. Apocynol [4-(1-hydroxyethyl)-2-methoxyphenol] was found to be the main degradation product, and its formation was shown to be highly dependent on the beer pH. Because both apocynol and vanillin have a clear vanilla-like aroma, the decrease of 4-vinylguaiacol during beer aging might impart a shift from a clove-like aroma in fresh specialty beers (such as wheat beers and other top-fermented blond or dark ales) to a sweeter, more vanilla-like flavor impression of aged specialty beers.
Volatile phenols are strong odorants produced by microbial activity and reported in several foods, but very scarce information is available on their presence in virgin olive oils (VOOs) and on their relation with VOO chemical and sensory quality. In the present paper, a factorial experimental design was applied for the development of a suitable solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) analytical method for the analysis of volatile phenols in olive oil. The memory effects demonstrated by SPME fibres required the optimization of desorption conditions to minimize experimental errors. A series of nine volatile phenols were identified and quantified for the first time in VOOs by analyzing samples with distinct off-flavours. Their limits of detection and quantification (microg/kg) were largely below the odour detection thresholds (ODTs) calculated in this study (mg/kg), confirming the capacity of the technique to assess the target compounds at early stages of the oil sensory alteration. The odour activity values (OAVs) of volatile phenols were calculated in VOOs facilitating a first assessment of their potential importance in the aroma of the product.
Sequence analysis of a Bacillus sp. BP-7 recombinant clone coding for a previously described carboxylesterase revealed the presence of an additional ORF with homology to bacterial hydroxycinnamic acid decarboxylases. Analysis of the amino acid sequence of the encoded enzyme revealed the presence of a single, highly conserved domain of 161 amino acids, with a predicted molecular mass of 19,143 Da and a pI of 5.5. Crude cell extracts from the recombinant clone displayed activity on ferulic, p-coumaric and caffeic acids, with no need for added cofactors. The cloned enzyme, named PadA, displayed maximum activity at 40 degrees C and pH 5.5, being stable over a broad range of pH and up to 45 degrees C. HPLC analysis of the products of catalysis revealed the conversion of phenolic acids to their aromatic 4-vinyl derivatives, with no accumulation of other by-products. PadA was found as a homodimer in the parental Bacillus sp. BP-7 strain and its expression was induced by both hydroxycinnamic acids and their corresponding derivative products. The results obtained suggest that the enzyme could be involved in a stress response for conversion of toxic hydroxycinnamic acids released after plant cell wall degradation.
The first Alicyclobacillus spp. was isolated in 1982, and was originally thought to be strictly limited to thermophilic and acidic environments. Two years later, another Alicyclobacillus sp., A. acidoterrestris, was identified as the causative agent in spoilage of commercially pasteurized apple juice. Subsequent studies soon found that Alicyclobacillus spp. are soilborne bacteria, and do not strictly require thermophilic and acidic environments. Alicyclobacillus spp. posess several distinct characteristics; the major one is their ability to survive commercial pasteurization processes and produce off-flavors in fruit juices. The fruit juice industry has acknowledged Alicyclobacillus spp. as a major quality control target microorganism. Guaiacol and halophenols were identified as the offensive smelling agent in many Alicyclobacillus spp. related spoilage. Though the exact formation pathway of these off-flavors by Alicyclobacillus spp. are not yet identified, studies report that the presence of Alicyclobacillus spp. in the medium may be a major contributor to the formation of these off-flavors. Many identification methods and isolation media were developed in the last two decades. However, most of these methods were developed specifically for A. acidoterrestris, which was the first identified off-flavor producing Alicyclobacillus. However, recent studies indicate that other species of Alicyclobacillus may also produce guaiacol or the halophenols. In this respect, all Alicyclobacillus spp. should be monitored as potential spoilage bacteria in fruit juices. This article includes an overall review of the history of Alicyclobacillus spp., characteristics, suggested off-flavor production pathways, and commonly used identification methods for the currently identified Alicyclobacillus spp.
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.
The suitability of a simple and rapid isocratic RP-HPLC method with amperometric electrochemical detection for the simultaneous detection and quantification of hydroxycinnamic acids and their corresponding aroma-active volatile phenols in wort and beer is reported. The technique gives good specificity and sensitivity, and can therefore be used for routine monitoring of hydroxycinnamic acids in wort and the development of volatile phenolic flavour compounds during the beer production process and subsequent conservation.
Yeasts were isolated from frozen concentrated orange juice, grown in Sabouraud dextrose broth at 25°C, and tested for the ability to cometabolize ferulic acid. Strains of Rhodotorula sp., Candida lambica, Trichosporon pullulons, and Candida intermedia decarboxylated ferulic acid nonoxidatively to an off-flavor compound, 4vinylguaiacol. By decarboxylating naturally occurring ferulic acid, these and other yeasts have the potential to contribute to off flavors in improperly stored fruit juices. Copyright © International Association of Milk, Food and Environmental Sanitarians.
Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.
A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.
Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.
A high-performance liquid chromatography (HPLC) analytical method, using a combination of ultraviolet-diode array (UV) and electrochemical (EC) detectors, was developed for the characterization and quantification of phenolic and furfural compounds in apple juice. The limits of detection of phenolic compounds with EC detection were 4–500 times greater than those obtained with UV detection. The HPLC method allowed the identification and quantification of the major (chlorogenic and coumaroylquinic acids and phloridzin) and minor (caffeic, p-coumaric, ferulic, gallic and protocatechuic acids, and catechin) phenolic compounds, as well as 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde in apple juice. The total phenolic content in apple juices of various origins varied from 28.85 to 115.49 mg/l. The concentrations of phenolic and furfural compounds varied widely among the non-concentrated and concentrated apple juices.
A novel plate assay method, developed for the screening of microorganisms or enzyme preparations for phenolic acid esterases, involves incorporating ethyl cinnamate into an agar medium. After inoculation and incubation, the plate is flooded with a pH-sensitive dye to reveal yellow zones around positive cultures against a blue background. A number of yeasts (Rhodotorula spp. and Candida spp.) and fungi (Penicillium sp. and Aspergillus sp.) gave positive results, while a number of commercial enzymes, particularly pectinases, also exhibited good phenolic acid esterase.
Seven isomeric dehydrodimers of ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been synthesized and identified in extracts of saponified cell walls of cocksfoot, switchgrass, and suspension-cultured corn. Dehydrodimers (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid, (Z)-beta-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxy-cinnamic acid, (E)-3-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-5-methoxycinnamic acid. (E,E)-4,4'-dihydroxy-3,5'-dimethoxy-beta,3'-bicinnamic acid, 4,4'-dihydroxy-3,3'-dimethoxy-beta,beta'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1,2-dihydro-naphthalene-2,3-dicarboxylic acid, all arise from oxidative coupling of ferulate esters in cell walls and represent products of 8-5, 8-8, 8-O-4, 4-O-5, and 5-5 radical coupling. Prior literature has acknowledged only the presence of the 5-5-coupled dehydrodimer (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid. Consequently, by measuring only a single dehydrodimer and assuming inappropriate response factors, ferulate dehydrodimers have been underestimated by factors of up to 20. Synthetic routes to all seven isomers have been developed to provide structural authentication and determination of GC response factors.
Purées and concentrates are intermediate products in the elaboration of commercial fruit juices. In this paper, the phenolic composition was established and quantified in a large number of samples of peach and apple purées and concentrates. Different benzoic acids and aldehydes, cinnamic acids and their derivatives, flavan-3-ols, procyanidins, flavonols, and dihydrochalcones have been identified. The concentration of cinnamic acids and derivatives is higher in the concentrates than in the purées for both fruits. However, flavan-3-ols and procyanidins are only present in the purées. Peach-based products are completely devoid of flavonol and dihydrochalcone derivatives due to the removal of the skin of the fruit in the manufacturing process. On the other hand, different quercetin and phoretin glycosides were detected in apple purées and concentrates. The results show that phenolic compounds can prove to be helpful in the characterization of fruit purées and concentrates as well as in the detection of adulterations in the manufacturing of commercial fruit juices from these intermediates.
HPLC techniques were used to study the effect of processing, concentration, and storage on the phenolic composition of Granny Smith apple juice pressed from fruit held at 1°C for 3 and 9 months. Extensive phenolic degradation was prevented by initial high-temperature short-time (HTST) treatment. Enzymatic clarification caused cinnamic hydrolysis and some procyanidin degradation. Fining and concentration had little effect on phenolics. Storage of concentrates for 9 months at 25°C resulted in (hydroxymethyl)-furfural (HMF) formation (up to 27.9 mg/L), degradation of cinnamics (ca. 36%) and quercetin and phloretin glycosides (ca. 60% ), and total loss of procyanidins. The effect of diffusion extraction at 55, 63, 67, and 73°C on the phenolic composition of Red Delicious, McIntosh, and Spartan apple juice was also studied. Up to a 3-fold increase in cinnamics and a 5-fold increase in phloretin glycosides was measured in diffusion-extracted juices relative to pressed juices. The effect of diffusion extraction on increased levels of procyanidins and phloretin and quercetin derivatives was even more pronounced.
High levels of 4-vinylphenol and 4-vinylguaiacol were detected in wines made from grape juice initially treated with some enzyme preparations. Two enzyme activities, which operate successively, are responsible for this formation. First, the cinnamoyl esterase activity from enzyme preparation liberates cinnamic acids from their corresponding tartaric acid esters. Second, cinnamic acids are transformed into 4-vinylphenol and 4-vinylguaiacol by decarboxylase activity provided by the yeasts. This activity is quite stable throughout alcoholic fermentation. The high levels of volatile phenols in some enzymatically treated wines could be responsible for unpleasant phenolic off-flavors. During storage, these compounds decreased and corresponding ethoxyethylphenols increased.
4-Vinylguaiacol (PVG), a major off-flavor in citrus products, was detected in stored model solutions of orange juice (MOJ) containing ferulic acid, and its amount increased with time and temperature. PVG was not found in MOJ incubated without ferulic acid. Vanillin, another ferulic acid degradation product, was also detected in MOJ containing ferulic acid after incubation at 35 and 45-degrees-C, but only minute amounts occurred at 25-degrees-C. Vanillin was not produced, however, in MOJ incubated with added PVG but which did not contain ferulic acid. Incubation of MOJ under nitrogen atmosphere rather than air or including butylated hydroxytoluene did not affect PVG levels even though nonenzymic browning products such as 5-(hydroxymethyl)furfural and furfural, and optical density values were reduced. Cu ions accelerated browning but decreased PVG levels. It appears that different factors affect PVG formation and sugar degradation.
The suppression of plant growth by different phenolic acids is well known. This work was designed to determine if ferulic acid, a known phenolic inhibitor of plant growth, accumulates in the soil and if soil microorganisms could be isolated that metabolize it. Over 99% of the extractable ferulic acid was lost from decaying hackberry leaves in 300 days. During this time the amount in the top 15 cm of soil remained fairly constant at about 30 ppm, except for the March sample which was significantly higher than the rest. Addition of ferulic acid to soil caused an increase in CO2 evolution and in numbers of a select group of microorganisms.Rhodotorula rubra andCepnalosporium curtipes, which actively metabolize ferulic acid, were isolated, but the metabolic pathways employed appear to be different from the reported one. The reported pathway for ferulic acid breakdown is ferulic acid to vanillic acid to protocatechuic acid to -keto-adipic acid.Rhodotorula Rubra was found to convert ferulic acid to vanillic acid, but no evidence was found for utilization of the rest of the pathway.Cephalosporium curtipes appears to use a different pathway or to metabolize intermediate compounds rapidly without accumulating them, because no phenolic compounds were found during the breakdown of ferulic acid. The presence in the soil of microorganisms that metabolize ferulic acid and other phenolic acids is ecologically significant because such organisms prevent long-term accumulations of these substances, which are toxic to many other microorganisms and higher plants.
Simple phenol and phenolic compounds occur in a vast portion of our diet. This review attempts to discuss their occurrences relative to our food supply. In addition, their chemical, nutritional, and sensory properties are explored, as well as methods for their isolation, identification, and quantitation.
A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.
The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids. Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation. The nucleotide sequence of the smallest complementing fragment was determined. The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli. PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype. PAD1 is a single-copy gene in the yeast genome and not essential for viability. The PAD1 locus was physically mapped to a position approx. 140 kb from the left end of chromosome IV.
Extracellular esterase production by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger was determined in both liquid and solid-state culture. Methyl ferulate was used as the main carbon source in liquid culture whereas wheat bran and sugar beet pulp were used in solid-state culture. Extracted enzyme for each fungus showed activity in the presence of ONP butyrate, methyl ferulate, methyl coumarate and two 'natural' feruloylated carbohydrate esters. Higher enzyme recoveries were obtained using wheat bran in solid-state culture. Higher levels of feruloyl esterase activity were recovered from P. expansum on all feruloylated substrates than from P. brevicompactum or A. niger. Using ONP butyrate as substrate the pH and temperature optima for the esterases of both Penicillium spp. were 6.0 and 25-30 degrees C. Aspergillus niger esterase activity showed a broader temperature range with an optimum at 40 degrees C.
In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin; O-demethylation; and reduction reactions. Biotransformations of vinylguaiacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.
The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agarplate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 degrees C respectively.