THE EVOLUTION OF AROMATIC PROFILE OF SANGIOVESE WINE DURING FERMENTATION AS REVEALED BY A QUANTITATIVE SPME-GCMS APPROACH: METHOD

DEVELOPEMENT

THE EVOLUTION OF AROMATIC PROFILE OF SANGIOVESE WINE DURING FERMENTATION AS REVEALED BY A QUANTITATIVE SPME-GCMS APPROACH: METHOD

DEVELOPEMENT

Luca Calamai1,2, Irene Lozzi 1, FedericoValori1, Francesca Boscaro2

1 Centro Interdipartimentale di Spettrometria di Massa, Università di Firenze 2 Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Università di Firenze

During winemaking process, the profile of aromatic compounds evolves and the primary varietal scent from the grapes are complemented and/or transformed by the yeasts’ activitey. Also, condensation, hydrolysis and trans-esterification reactions take places recurrently as the concentration of alcools and organic acids continuously change during wine making catalyzed by must and wine acidic pH .

-Both primary and secondary compounds are revealed by SPME-GCMS analysis

-Triphasic DVB/CARBOXEN/ PDMS fiber afforded the best performances

-Reliable quantation curves were obtained by the use of a multiple ISTD mix composed of labelled compounds

CONCLUSIONS

Experimental-Samples collected 1 day after crushing and destemming- Different wine making treatments:

Cold prefermentation maceration at 5 °CCrio macetation with liquid nitrogenTraditional winemaking

Is it possible to gather quantitative information on the volatile compound during wine making by SPME- GCMS as a function of different oenological tretments to be used as feedback process indicators?

Method developement-5 ml of must or wine supplemented with 2 g of NaCl in 20 ml vials-5 types ofSPME fiber: DVB/CARBOXEN/ PDMS fiber,- sorption times, f rom 5 , 10, 30 min min-Addition of labeled internal standard mix to normalize responses.-column J&W Innowax 30 m, 0.25 mm, ID 0.5 µm DF; injection temperature 250°C, splitless mode, oven program: 40 degrees for 1 min then 2°C/min to 60 °C, then 3°C/min to 150°C, then 10°C/min to 200°C, then 25°C/min to 260°C for 6.6 min

Agilent 5975C MSD spectrometer with Gerstel MPS2 XL equipped with SPME automatic fiber exchange option

0

Acetic acid istd

Response Ratio

Concentration Ratio

R2=0.9952

R2=0.7090

2000 3000

acetic acid ext stdResponse

0

R2=0.9849

4-ethylphenol ext stdResponse

Concentration (ppm)

0 2 4

0 2 4

4-ethylphenol istdResponse Ratio

Concentration Ratio

R2= 0.9998

Concentration (ppm)

1000

2000 30001000

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.000

Time

Ab

un

dan

ce

CARBOWAX_DVB

CARBOXEN_PDMS

PDMS

PDMS_DVB

TRIPHASE (DVB/PDMS/Carboxen)

Ph

en

yle

thyl alc

oh

ol

Ben

zyl alc

oh

ol

Cap

roic

acid

D1

1 (

ISTD

6)

Eth

an

ol,

2-(

2-e

thoxyeth

oxy)-

Aceti

c a

cid

5-

meth

yl h

exan

ol (I

STD

4)

2-B

uta

non

e,

3-h

yd

roxy-

eth

yl h

exan

oate

D1

1 (

ISTD

3)

am

yl+

isoam

yl alc

oh

ol

Bu

tan

ol D

10

(IS

TD

2)

1-P

rop

an

ol,

2-m

eth

yl-

Eth

yl aceta

te D

8 (

Istd

1)

Meth

an

ol

Eth

an

ol

Aceti

c a

cid

D3

(IS

TD

5)

Hexad

ecan

oic

acid

Dib

uty

l p

hth

ala

te

Tetr

ad

ecan

oic

acid

Bu

tyl p

hth

ala

te

Ph

en

ol,

2,4

-bis

(1,1

-dim

eth

yle

n-d

ecan

oic

acid

3-4

dim

eth

ylp

hen

ol (I

STD

7)

Non

an

oic

acid

BH

T

Octa

noic

acid

Hexan

ol

Cap

roic

acid

The best overall results were obtained with a 65 um carboxen/PDMS/ DVB SPME fiber exposed in the head space of a 20 ml vial under continuous agitation for 20 min at 80°C

Reliable calibration lines were obtained when the appropriate internal standards (ISTD) was used for calibration. An ISTD mix composed of two compounds (fast and late eluting) for each class was added to all samples.

Canonical Scores Plot

TESTMPFCRIO

TREATMENT

-9 -1 7 15FACTOR(1)

-9

-1

7

15

FA

CT

OR

(2)

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

Abu

ndan

ce

TIC: must after 1 day

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.00 50.0 55.0

0

Time

TIC: wine after racking

Bu

tan

oic

acid

, eth

yl este

r

1,1

die

thoxyeth

an

eeth

yl aceta

teEth

yl aceta

te D

8 (

Istd

1)

Hexad

ecan

oic

acic

id

Tetr

ad

ecan

oic

oti

c a

cid

Dod

ecan

oic

acid

3,4

-dim

eth

yp

hen

ol (i

std

7)

Ben

zoic

acid

eth

yl octa

note

1-D

od

ecan

ol

eth

yl d

ecan

oate

ß-d

am

ascen

on

e ß-d

am

ascen

on

e t

ic

Cap

roic

acid

Ben

zald

eh

yd

e,

3,4

-dim

eth

yl ti

c

Meth

yl d

can

oate

lin

alo

l

ben

zald

eid

e

aceti

c a

cid

Aceti

c a

cid

D3

(IS

TD

5)

5-

meth

yl h

exan

ol (I

STD

4)

Eth

yl h

exan

oate

D1

1 (

ISTD

3)

1-b

uta

nol

bu

tan

ol D

8 (

ISTD

2)

1-B

uta

nol,

3-m

eth

yl-

, aceta

te

1-P

rop

an

ol,

2-m

eth

yl-

1-p

rop

an

ol

Pro

pan

oic

acid

, eth

yl este

r

eth

yl h

exan

oate

Am

yl +

isoam

yl alc

oh

ol

1-h

exan

ol

Aceto

in

2,3

bu

tan

ed

ion

e

2,3

bu

tan

ed

iol C

ap

roic

acid

D1

1 (

ISTD

6)

Ph

en

yle

thyl alc

oh

ol

dod

ecan

oic

oti

c a

cid

eth

yl d

od

ecan

oate

octa

noic

acid

Eth

yl la

cta

te

Discriminant analysis on compound concentration indicate statistically significant grouping with treatments

almost all identified compounds are included in the composition of discriminant factorsBoth primary and secondary aromatic

compounds are identified in the samples. SPME-GC ms analysis during winemaking may provide insights on the fermentation dynamics

Canonical Discriminant Functions : Standardized by Within Variances

1 21_1_DIETHOXYETHANE 0.36309 5.047257EHYL_ACETATE 4.780526 -1.35864

PROPANOIC_ACID_ETHYL_ESTER -4.77559 -0.71234

BUTANOIC_ACID_ETHYL_ESTER -4.00784 1.030564

1_BUTANOLO 0.29489 0.3683571_PROPANOL_METHYL_ 2.531798 -1.33972AMYL +_ISOAMYL ALCOHOL -3.66267 -13.293

1_BUTANOL_2_METHYL_ACETATE 1.294225 10.1613

ETHYL_HEXANOATE -3.69002 -1.28373ETHYL_LACTATE 3.436858 2.949362ETHYL_DECANOATE 1.805871 4.620106ETHYL_DODECANOATE -2.03198 -2.56303ETHYL_TETRADECANOATE -6.30733 -2.145021_HEXANOL -3.18469 -2.569372_HEXENAL-E -0.36237 -1.845383_HEXEN_1_OL_Z -1.23956 -0.663181_OCTANOL 2.98669 0.7728861_DODECANOL -2.2893 -2.27307OCTANOIC ACID 0.397371 14.59236N_DODECANOIC ACID -3.68283 1.045351N_DECANOIC ACID 15.40138 -10.0614BENZOIC ACID -5.3553 -4.00649TETRADECANOIC TIC 4.790019 0.504301HEXADECANOIC ACID . .GERANIOL -2.33336 -1.16235LINALOL . .BENZALDEHYDE -0.39774 -1.26008BENZALDEHYDE _4_ETHYL 1.443666 2.239546BENZYL_ALCOHOL 1.017217 1.036393PHENYLETHYL_ALCOHOL . .PHENOL_4_BIS_1_1_DIMETILETHYL

-2.12442 -4.53027