Trans caftaric acid

External standards for 1-kestose, nystose, raffinose, glucose, fructose, rhamnose, maltose and for citric tartaric, lactic and acetic acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). External standards for formic and succinic acids were purchased from Merck (Darmstadt, HE, Germany). External standards for procyanidin A2, epigallocatechin gallate, epicatechin gallate, kaempferol 3-glucoside, quercetin 3-rutinoside (rutin), quercetin 3-glucoside, myricetin, came from Extrasynthese (Genay, France). cis-Resveratrol and trans-resveratrol were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). External standards of the phenolics: gallic acid, p-coumaric acid, chlorogenic acid, syringic acid, trans-caftaric acid, caffeic acid, hesperidin, naringenin, procyanidin B1, catechin, epicatechin and procyanidin B2, were purchased from Sigma-Aldrich. Acetonitrile, methanol, potassium persulfate, gallic acid and ethyl alcohol were obtained from Merck. The ultrapure water was obtained using a Marte Científica purification system (São Paulo, SP, Brazil). The chromogen 3,3′diaminobenzidine tetrachloride was purchased from Vector Laboratories (Burlingame, CA, USA). All antibodies used in this study were purchased from Abcam (Cambridge, MA, UK). All chemical reagents used in the experiments were of analytical grade.

Acetonitrile and formic acid, HPLC quality, were supplied by Labscan (Dublin, Ireland) and Acros Organic (Belgium), respectively. Protocatechuic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, 3-coumaric acid, ethyl gallate, 3,5,4’-trihydroxystilbene-3-O-β-d-glucoside (trans-piceid), (+)-catechin, (−)-epicatechin, epicatechin gallate, procyanidin B₁, procyanidin B₂, procyanidin B₃, quercetin-3-O-galactoside, quercetin-3-O-rutinoside, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, quercetin dihydrate, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside and malvidin-3-O-glucoside were purchased from Extrasynthèse (Genay, France). Gallic acid, 4-hydroxybenzoic acid, trans-caftaric acid, trans-ferulic acid, ellagic acid, protocatechuic aldehyde, trans-resveratrol, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), potassium persulfate, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 1 M phosphate buffer, fluorescein sodium and 2,2’-azobis(2-methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma-Aldrich (Madrid, Spain). Disodium carbonate, Folin–Ciocalteu reagent, methanol and ethanol were from Panreac (Barcelona, Spain).

In order to study whether the cinnamoyl esterase activity in O. oeni is somehow related to a detoxification effect, the toxic levels of trans-caftaric acid and its immediate phenolic metabolites (trans-caffeic acid and 4-ethylcatechol) were evaluated in MRS broth supplemented with ethanol and cycloheximide (at 5% v/v and 10 mg/L, respectively) and with pH adjusted to 4.5.
Solutions of 4-ethylcatechol (4EC – purity 95%), trans-caftaric and trans-caffeic acids (Sigma-Aldrich, Germany) were prepared immediately before use from the pure compounds by dissolving the appropriate amounts in ethanol (99.5% v/v). Fresh cultures of the five O. oeni strains from Ch. Hansen were cultivated in 96-well × 300 μL microplates containing MRS broth supplemented with 300 mg/L and 150 mg/L of trans-caftaric acid, 170 mg/L and 85 mg/L of trans-caffeic acid and 130 mg/L and 65 mg/L of 4EC, representing equivalent molar concentrations (1 and 0.5 mmol/L respectively) for all compounds. Cultures were grown at 25°C with no agitation for 9 days with 1 day as interval of absorbance measurement. Absorbance was measured (at 600 nm wavelength) with a Synergy^TM HTX Multi-Mode Microplate Reader from BioTek Instruments (Winooski, VT, United States).