Vitis 41 (2), 107112 (2002) Changes in carbonyl compounds in Chardonnay and Cabernet Sauvignon wines as a consequence of malolactic fermentation R. F LAMINI 1) , G. D E L UCA and R. D I S TEFANO 2) 1) Istituto Sperimentale per la Viticoltura, Conegliano (TV), Italia 2) Istituto Sperimentale per lEnologia, Asti, Italia Summary attack on berries. The three above mentioned compounds are probably very important for wine flavor, because of their To study changes in carbonyl compounds in Chardonnay very low odor threshold. Moreover, dicarbonyl compounds and Cabernet Sauvignon wines as a consequence of malol- are associated with browning processes due to their reac- actic fermentation (MLF), wines were fermented by inocu- tions with amino acids; they are more toxic than their reduc- lation of commercial strains of Oenococcus oeni , and com- tion products ( DE R EVEL and B ERTRAND 1993 a, G UILLOU et al . pared with unfermented (control) wines. Carbonyl com- 1997). pounds were determined by GC/MS analysis on the basis of In this work, we studied changes in carbonyl compounds their O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine deriva- as a consequence of MLF in Chardonnay and Cabernet tives after sample preparation on an ion exchange column Sauvignon wines. We performed fermentation by inocula- to remove pyruvic acid. With MLF, marked changes were tion of two commercial Oenococcus oeni strains; the carbo- revealed, particularly with regard to diacetyl, acetoin and nyl compounds of the wines were compared with those of aliphatic saturated aldehydes; the presence of unsaturated unfermented controls. Compounds were determined by GC/ aldehydes was also revealed. A significant increase in MS analysis of their O-(2,3,4,5,6-pentafluorobenzyl)-hy- glycoladehyde was observed, which is presumed to be part droxylamine (PFBOA) derivatives ( DE R EVEL and B ERTRAND of a reduction system with glyoxal. Higher acetoin/diacetyl 1993 b). ratios were found in Chardonnay and higher glycolalde- hyde/glyoxal ratios in Cabernet Sauvignon. Material and Methods K e y w o r d s : carbonyl compounds, glycolaldehyde, malolactic fermentation, wine, PFBOA ( O-(2,3,4,5,6- W i n e s : Cabernet Sauvignon and Chardonnay wines pentafluorobenzyl)-hydroxylamine), GC/MS. were produced from grapes cultivated in Conegliano (Veneto, Italy) and harvested in 2000. Must was fermented at the Istituto Sperimentale per la Viticoltura of Conegliano. At the Introduction end of fermentation, sugar residues in wine were less than 2 g ×l-1 . In order to limit carbonyl/bisulfite adduct formation During malolactic fermentation (MLF) also carbonyl and to eliminate the basic hydrolysis step in sample prepa- compounds undergo profound changes, by which the orga- ration, no SO 2 was added during or after alcoholic fermenta- noleptic characteristics of the wine can be improved tion. This also favored bacterial growth (N IELSEN and (S AUVAGEOT and V IVIER 1997). Changes in the herbaceous RICHELIEU 1999). odor of wine were observed with MLF, associated with M a l o l a c t i c f e r m e n t a t i o n : MLF was performed methoxypyrazines, some C6 alcohols, and some aliphatic by membrane-resistant (MBR) Uvaferm ALPHA (strain A) aldehydes such as hexanal, ( E)-2-hexanal, ( E)-2-eptenal, and MLD (strain B) (Esseco spa, Trecate, Italy) according to octanal, ( E)-2-octanal ( DE R EVEL and B ERTRAND 1993 a, A LLEN the manufacturers guidelines. Lyophilized bacteria (50 mg) 1995). Changes in decanal and ( E)-2-nonenal contents, as- were hydrated in 2 ml of distilled water at 25 °C for 15 min. sociated with sawdust or plank odor (C HATONNET and The bacterial suspension was added to 200 ml of wine, stirred DUBOURDIEU 1996, 1998), may also occur. for 1 h and kept at 25 °C for about 10 d. Fermentation was During MLF, the butter-like fat note increases, due to followed by organic acid screening (until the disappearance 2,3-butanedione (diacetyl) and its reduced form, 3-hydroxy- of malic acid) by HPLC analysis (F LAMINI and D ALLA V EDOVA 2-butanone (acetoin). The second reduced form, 2,3-butane- 1999). Organic acids, pH and titratable acidity of samples diol, is linked to the cooked note (D AVIS et al . 1985, DE R EVEL before and at the end of MLF are listed in Tab. 1. and B ERTRAND 1993 a, L AURENT et al . 1994). The HPLC system consisted of a chromatograph Varian Carbonyl compounds glyoxal, methylglyoxal and 9010 (Varian Instrument, Walnut Creek, CA, USA) equipped hydroxypropandial are also present in wine. They are pro- with a 20 µl loop, connected to a Varian 2550 UV-vis detector duced by microorganisms such as Saccharomyces (wavelength 210 nm). Column: LiChrospher 100 RP-18 cerevisiae and Leuconostoc oenos , and by Botrytis cinerea (250 mm x 4.6 mm i.d., 5 µm) with LiChrocart guard column Correspondence to: Dr. R. F LAMINI , Istituto Sperimentale per la Viticoltura, Viale XXVIII Aprile 26, I-31015 Conegliano (TV), Italy. Fax: +39-438-450773. E-mail: [email protected] 108 R. F LAMINI , G. D E L UCA and R. D I S TEFANO T a b l e 1 Organic acids, titratable acidity and pH of wines before MLF (control) and after MLF by two Oenococcus oeni strains Cabernet Sauvignon Chardonnay control strain A strain B control strain A strain B malic acid (g·l -1 ) 1.81 trace trace 2.53 trace trace lactic acid (g·l -1 ) 0.54 1.94 1.94 0.10 1.71 1.72 pyruvic acid (g·l -1 ) 0.14 0.01 0.01 0.08 0.05 0.05 acetic acid (g·l -1 ) 0.25 0.43 0.47 0.44 0.47 0.48 titratable acidity (g·l -1 ) 7.90 6.80 6.80 7.60 5.15 5.15 pH 3.55 3.70 3.70 3.24 3.50 3.50 (4 mm x 4 mm, 5 µm) (E. Merck, Darmstadt, Germany). A Varian ethanol. The first was prepared by diluting 0.5 ml of stand- 4400 integrator connected with WOW Chemstation software ard solution in 50 ml ethanol/water (12 % v/v, pH 3), fol- (Thermo Separation Products, Riviera Beach, FL, USA) was lowed by addition of 10 ml ethanol and 200 ml internal stand- used. Fermentations were carried out in duplicate. ard. Reaction and analysis were performed as reported above. R e a g e n t s a n d s t a n d a r d s : O -(2,3,4,5,6- The second sample was prepared by diluting 0.5 ml of stand- penta-fluorobenzyl)-hydroxylamine hydrochloride ard solution in 50 ml ethanol/water (12 % v/v, pH 7.5) and (PFBOA), decanal, ( E)-2-hexenal, ( E)-2-nonenal, nonanal, then passing it through a preconditioned ion exchange col- diacetyl, ( E,E )-2,6-nonadienal, ( E,Z )-2,6-nonadienal, ( E)- umn (Amberlite IRA 400 10 g). The column was washed with 2-pentenal, ( E)-2-octenal, ( E)-2-heptenal and glycoladehyde 10 ml ethanol, the organic phase combined with the aque- were purchased from Sigma-Aldrich (Milan, Italy). Vanil- ous one, and pH was adjusted to 3. Internal standard was lin, acetoin, pentanal and glyoxal were purchased from Fluka added to the solution; reaction and analysis were performed (Milan, Italy); ( E)-crotonaldehyde and heptanal from Carlo as reported above. The recovery of each compound from Erba Reagenti (Milan, Italy); hexanal from Merck (Milan, the column was estimated on the basis of the difference Italy) and acetaldehyde from BDH. between peak areas in the chromatograms of the two sam- S a m p l e p r e p a r a t i o n f o r c a r b o n y l ples. Experiments were repeated three times. c o m p o u n d a n a l y s i s : The ion exchange column was S a m p l e p r e p a r a t i o n f o r 2 , 3 - b u t a n e d i o l prepared with 10 g of Amberlite IRA 400 resin (Fluka, Milan, a n a l y s i s : Samples were prepared with Isolute ENV(+) Italy), preconditioned with 20 ml of HCl 1 % (v/v), 100 ml 500 mg cartridges (International Sorbent Technology Ltd., NaF 0.5 mol and rinsed with water. The pH of 50 ml of wine UK), preconditioned by the passage of 10 ml methyl chlo- was raised to 7.5 by the addition of 1 ml NaOH (4 mol). This ride and 10 ml methanol, rinsed with 20 ml water. 15 ml of solution was passed through the column. The stationary wine was adjusted to 45 ml with water, and 250 ml of a phase was washed with 10 ml ethanol and both fractions 1-heptanol (1.53 mmol) solution were added as internal stand- passed through the column were combined. After the addi- ard. The solution was loaded on the cartridge, the station- tion of 200 ml o-chlorobenzaldehyde (0.105 mmol) as internal ary phase raised with 20 ml water, and the organic fraction standard, the solution was adjusted to pH 3.0; after the ad- eluted with 8 ml of methyl chloride. The organic phase was dition of 20 mg PFBOA, it was stirred for 1 h at room tem- dried over Na 2SO 4 and filtered. The volume was reduced to perature. O-pentafluorobenzyl oximes ( O-PFB-oximes) were 0.4 ml before performing GC/MS analysis (SCAN mode).