Bioconversion de l'acide p-coumarique par Brettanomyces ...

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Author's personal copyD. Salameh et al. / Food Chemistry 107 (2008) 1661–1667 1663The automatic injector was used in a full loop of 20 ll,with a mobile phase composed of: water 77%, acetonitrile23%. Formic acid was added in the concentrationof 0.12 g/l to regulate the pH at 3.5. The flow was fixedat 0.7 ml/min. The detector used was a UV spectrophotometer.The detection was done at 305 nm. p-Coumaricacid retention time was 12.5 min.2.2. Stability tests of the p-coumaric acid with experimentalconditionsIn order to study the day light and oxygen effect onthe p-coumaric acid in our experimental conditions, two40 mg/l solutions of p-coumaric acid were prepared inErlenmeyer flasks of 500 ml containing 300 ml of liquid.The first solution was a synthetic wine medium with10% v/v ethanol; the second was a hydroalcoholic solutioncontaining distilled water with 10% v/v ethanol.Both of the solutions were stirred for 10 days at ambienttemperature, closed inside the Erlenmeyer flasks. Eachsolution was monitored by UV spectrophotometry at305 nm.On the other hand, to study the p-coumaric acid thermodegradationat high temperature, two 40 mg/l solutionsof p-coumaric acid dissolved into a synthetic wine mediumwere prepared into 50 ml volume Pyrex Shott containers.They were kept in darkness without any contact with air.Two temperatures were tested: ambient temperature(approximately 20 °C) and 75 °C obtained using a heatingplate. The changes in absorbance at 305 nm were detectedin time. Absorbance changes of the p-coumaric acid solutionwere equally tested before and after autoclaving for20 min at 120 °C.2.3. Reactivity tests of the p-coumaric acid with mediacomponentsTo study the p-coumaric acid dissolution in ethanol,10 mg/l of the acid were added either after being dissolvedor not into ethanol to synthetic wine media containing 10%v/v ethanol. The absorbance of the acid was followed up intime by UV spectrophotometer in quartz cells at 305 nmfor 120 h.On the other hand, p-coumaric acid was either beingadded or not to different media to study its reactivity withethanol as a solvent or with tartaric acid. These differentmedia had the following composition: Medium A (Hydroalcoholic:water/ethanol 10% v/v), Medium B (water–20mg/l of p-coumaric acid), Medium C (pure ethanol–20 mg/l of p-coumaric acid), Medium D (Hydroalcoholic:water/ethanol 10% v/v–20 mg/l of p-coumaric acid), MediumE (synthetic wine medium 10% v/v ethanol), MediumF (synthetic wine medium 10% v/v ethanol–20 mg/l ofp-coumaric acid), Medium G (tartaric acid solution–20 mg/l of p-coumaric acid). The solutions were followedup by UV analysis with a sweeping wavelength between250 and 350 nm.2.4. Nuclear magnetic resonance (NMR) and infra red (IR)analysisTwo solutions of p-coumaric acid were prepared andtested by NMR to know whether a reaction occurs ornot between p-coumaric acid and tartaric acid or betweenp-coumaric acid and ethanol. The first contained 10 mg/lof p-coumaric acid dissolved in ethanol; the second contained10 mg/l of p-coumaric acid dissolved in tartaric acidsolution. In both solutions, pH was adjusted to 3.5 as inour oenological conditions. The NMR spectrum of thecrystalline p-coumaric powder was done after dissolutionin DMSO at 10 mg/l concentration in order to be comparedto other spectra. The NMR tests were recorded at300 MHz on a Brucker spectrometer. Chemical shifts areexpressed in part per million relative to tetramethylsilane.NMR solvent was pure DMSO.On the other hand, infra red analysis of the pure p-coumaricacid and the one dissolved into ethanol, where thepH was regulated at 3.5, were acquired on a Shimadzu-8300 Fourier Transform infra red spectrophotometer.2.5. Adsorption test of p-coumaric acid on BrettanomycesyeastsOne strain of yeast was isolated from Buzet winery, situatedin the south west of France. It was identified geneticallyas Brettanomyces bruxellensis. The Brettanomycesyeast was scraped from a Petri dish where it was storedat 4 °C.The yeast culture medium was a synthetic wine mediumthat had the following composition: glucose 20 g/l, yeastextract 0.5 g/l, (NH 4 ) 2 SO 4 0.5 g/l, citric acid 0.3 g/l, malicacid 3 g/l, tartaric acid 2 g/l, MgSO 4 0.4 g/l, KH 2 PO 4 5g/l, glycerol 6 g/l. Ethanol was added up to 10% v/v afterautoclaving. Culture of Brettanomyces was inoculated at3.10 6 cell/ml yeast concentration. Culture was carried outin 500 ml Erlenmeyer flask with 300 ml of liquid mediumstirred at 250 rpm. After inoculation, culture flasks wereincubated at 30 °C.Fermentation was carried out in order to have differentpopulation concentration of Brettanomyces yeast at differentstages of yeast growth.p-Coumaric acid was added in the fermentationdescribed above after dissolution in 1 ml of pure ethanol,to have respectively the concentration of 5, 10 or 20 mg/l.Two minutes after adding p-coumaric acid in each solution,a sample was taken, centrifuged, and then the liquid partwas tested on HPLC to detect the p-coumaric acid concentration,and on GC/MS to detect the vinylphenol and ethylphenolscontent. Three synthetic wine mediums (T1, T2and T3) free of Brettanomyces yeast were prepared containing,respectively, 5, 10 and 20 mg/l of p-coumaric acid and
Author's personal copy1664 D. Salameh et al. / Food Chemistry 107 (2008) 1661–1667served as a witness test. All these experiments were done ata temperature of 30 °C.2.6. Yeast countingBiomass concentration was followed up by two analyticaltechniques. A correlation between the optical density ofyeast suspension at 620 nm and cell dry weight gives weightconcentration, and the use of a Thoma hemacytometergives a concentration in number of cells. The percentageof viable cells is obtained using the ethylene blue stainingmethod, with an error of 8%.3. Results and discussion3.1. p-Coumaric acid stability in the air oxygen under daylightThe results showed that there was no significant changein absorbance after 10 days. It seems that the p-coumaricacid is stable in daylight, at ambient temperature and inthe presence of oxygen under experimental conditions.3.2. Thermodegradation test of p-coumaric acidThe purpose of this test was to study the stability of thep-coumaric acid with temperature. The results showed thatthere was no change in UV absorbance during 30 h underambient temperature, while the absorbance decreased whenthe temperature was 75 °C (Fig. 1). The absorbancedecreased by 26% (from 0.65 down to 0.481) when the p-coumaric acid solution was autoclaved. As a control, theabsorbance of a wine synthetic medium, free from the acid,was followed up before and after autoclaving. The absorbanceremained constant: 0.3 abs value.These tests showed that p-coumaric acid in solutions,particularly in natural wine under oenological conditions,is quite stable at ambient temperature. On the other hand,it is unstable when temperature increases. We confirm theresults of Bagchi et al., 1997 and Herrera et al., 1998. Practically,it would be important to add the p-coumaric acid insynthetic wine media after autoclaving in order to be surethat all the p-coumaric acid will be available in its naturalform for enzymatic reactions.3.3. Reactivity tests of the p-coumaric acid in the mediaIt is difficult to dissolve p-coumaric crystalline powder inpure distilled water. When it is dissolved, the p-coumaricacid takes filamentous form within 40 h. Authors proposedin their work the dissolution of p-coumaric acid in ethanolwhich is a potent solvent. On the other hand, Tuzen andOzdemir (2003) proposed the dissolution of p-coumaricacid into hydroalcoholic solutions.The observations showed that p-coumaric acid dissolvesslower in hydroalcoholic solutions than in pure ethanolsolution (Fig. 2). But they showed also that there was a lossof p-coumaric acid quantity relative to 0.2 absorbance unit,when dissolving into pure ethanol, which leads to thinkthat a probable ethanol/p-coumaric acid reaction couldtake place. The study of p-coumaric acid UV absorbancein the media detailed in the material and methods part(Media A–F), confirmed this hypothesis. It gave the followingresults.In the range studied, Medium A showed no absorption.Medium B showed two absorption bands at 285 nm and305 nm. On the other hand, media C and D showedremarkable hypsochrome and hypochrome effect. Themaximum wavelength absorption decreased from 284 nm(Medium B) to 248 nm (Medium C), and the band becamelarger.The hypochrome effect could result from a destabilizationof the mesomeric system which minimizes the absorptionprobability of the carbonyl group. The hypsochromeeffect could result even from the polarity change of the solventor from any change in the p-coumaric acid structure.Moreover, this peak shift occurred more slowly (24 h) inMedium D, where the medium had a little ethanol concentration(10% v/v ethanol). These observations lead to thinkthat some reactions between the p-coumaric acid and theethanol might take place and that the concentration of ethanolparticipates in its kinetics.0.850.8Absorbance0.70.680.660.640.620.60.580.560.540.520 5 10 15 20 25 30Time (h)Fig. 1. Absorbance as a function of time at ambient temperature 20 °C()and 75 °C (h) in synthetic wine medium.Absorbance0.750.70.650.60.550.50.450.40 20 40 60 80 100Time (h)Fig. 2. Kinetic profile of the dissolution of p-coumaric acid in ethanol ()and synthetic wine medium (h).
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Bioconversion de l'acide p-coumarique par Brettanomyces ...

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