Micro-oxygenation and its impact on
polyphenols and sensory characteristics
of red wines
Dominik Durner1,2, Patrick Nickolaus1, and Hai-Linh Trieu1
1 Dienstleistungszentrum
Ländlicher Raum (DLR) Rheinpfalz, Department of Viticulture &
Enology, Breitenweg 71, 67435 Neustadt an der Weinstraße, Germany
2 Hochschule
Kaiserslautern, Department of Applied Logistics and Polymer Sciences ,
Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 1
Viticulture in Germany
Source: Pfalzwein e.V.
Neustadt
Source: DWI
ASVO Seminar Inputs to Outputs; is less, more?
Source: Weingut Stephan Fischer
November 18th, 2014
Slide 2
DLR Rheinpfalz - State Research and Education
Center for Viticulture and Horticulture
© DLR Rheinpfalz
ASVO Seminar Inputs to Outputs; is less, more?
© DLR Rheinpfalz
© DLR Rheinpfalz
November 18th, 2014
©Slide
DLR3 Rheinpfalz
Which one is your money on?
2013 Pinot noir (pre bottling)
--------20 % Botrytis--------No Mox
Post-MLF Mox
(5/12wk)
Pre-MLF Mox
(20/3wk)
No Mox
Post-MLF Mox
(5/12wk)
Picture by Marlene Neser
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 4
Evolution of anthocyanins and tannins
during fermentation
2003 Pinot noir; 50 hL fermentation on skins (28°C/8 d); punch downs (4-12-4 per day)
Percentage of max. conetration
120%
100%
80%
60%
40%
Alcohol (89.4 g/L)
20%
Anthocyanins (182 mg/L)
Tannins (103 mg/L)
0%
0
1
2
Markus Löchner (2004)
ASVO Seminar Inputs to Outputs; is less, more?
3
4
5
Time (days)
6
7
November 18th, 2014
8
9
Slide 5
Evolution of colour from must to bottle
2012 Pinot noir; thermovinification (85°C/2 min); 3 hL juice fermentation (20°C/10 d); MLF
(18°C/35 d); micro-ox (10 mg/L/m/12 wk); 90/35 mg/L total/free SO2; bottle storage (15°C/9
m)
300
14
Anthocyanins (mg/L)
12
Colour Density (AU)
10
200
8
150
6
100
4
50
0
2
Pre AF
AF
(3 d)
Post AF
(10 d)
Pre MLF
(2 m)
Post MLF Post MOX
(3 m)
(6 m)
SO2
(7 m)
Bottling
(9 m)
3 m post-b 9 m post-b
(12 m)
(18 m)
Julia Willeke (2014)
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 6
0
Color Density (AU)
Anthocyanins (mg/L)
250
Reasons for the loss of anthocyanins
1. Polymerisation/derivatisation reactions  nice red pigments
 precipitation of polym. pigments
3. Adsorption to yeast cells or
fining agents  loss in colour
4. ß-Glucosidase-catalyzed
cleavage  low solubility
 loss in colour
Pinot noir
from vintages
2013, 2010,
and 1979
Remaining anthocyanins at bottling (%)
2. PPO catalysed oxidation
of anthocyanins  brown
pigments or colourless adducts
60
DV 10
IOC
50
40
30
20
10
0
Picture by Patrick Nickolaus
ASVO Seminar Inputs to Outputs; is less, more?
Julia Willeke (2014)
November 18th, 2014
Slide 7
Micro-oxygenation
Pros
Cons
• Stable colour pigments
• Risk of over-dosing
• Early drinking maturity
• High SO2 demand
• No reductive flavour
• Oxidised aroma
• Higher fruit intensity
• Dry tannins
• Less green tannins
• Stuck MLF
• Balanced tannins
• Fast aging
• Microbial safety
• Browning
• Less CO2
• No effect
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 8
Passive supply of oxygen
Barrels
1
HDPE-tanks
Synthetic cylindricals
New 225 L barrel:
3-4 mg O2/L/month1
5000 L HDPE-tank:
0.4 mg O2/L/month2
Low OTR:
0.01 mg O2/L/month3
Used 225 L barrel:
1-2 mg O2/L/month1
300 L HDPE-tank:
2.1 mg O2/L/month2
High OTR:
0.7 mg O2/L/month3
from Vivas (1997); 2 Flecknoe-Brown (2005); 3 product information Nomacorc Select Series
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 9
Active supply of oxygen (micro-ox)
Micro-ox
unit
O2
0.1 bis 180 mg O2/L/month
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 10
Technical prerequisites
Dissolved oxygen (µg/L)
0
20
40
60
80 100
0.1 m
Depth from wine surface
• Exact und continuous dosage of oxygen
• Production of small bubbles
 Slow ascent velocity of bubbles
• No sticking bubbles to diffuser
 Growing bubbles
• Quick dissolution of oxygen in the wine
 no accumulation of oxygen
• Good distribution of oxygen in the tank
1m
2m
3m
Diffuser at 3.5 m; n=3
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 11
Oxygen-induced formation of acetaldehyde
•
Semiquinone
Catechol
Semiquinone
Fe2+
Fe3+
•
Fe3+
HO2•
H 2 O2
Fe2+
OH •
Fe 3+
Formation of
oxygen radicals
Fe 2+
O2
CH3CH2OH
Ethanol
Fenton-Reaction
Quinone
CH3CHO
H2O
Acetaldehyde
adapted from Danilewicz (2003) Am J Enol Vitic 54: 73-85.
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 12
Acetaldehyde-induced reactions of
anthocyanins
OCH3
OH
Anthocyan (Mv)
HO
O
+
OCH3
OGlc
HO
OH
OH
Catechin (Cat)
OH
O
+
+ Acetaldehyde
+ Acetaldehyde
OH
OH
R
OH
GlcO
H3 CO
O
+
OH
OH
OH
HO
Vitisin B (R=H)
OCH3 H O
Mv-mm-Cat
O
OH
H
OH
adapted from Fulcrand (1998) Phytochem 47: 1401; Timberlake et al. (1976) Am J Enol Vitic 27: 97.
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 13
Winemaking protocol
2013 Pinot noir
Vinyard
Oberderdingen (Württemberg)
Harvest
Hand-picked on October, 12th, 2013
Gravity
20.9°Bx
pH
3.2
YAN
240
Chaptalising
110 g/L pot. Alc.
Winemaking
Temperature (AF)
28°C
Punch downs
4-12-4 per day (1 min)
Yeast
Mycoferm Cru 05
Simultanious MLF
Extremo IT14
Pressing
after 8 days
Temperature (MLF)
20°C
Racking
after 36 days
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 14
Micro-ox experiment
Height: 3 m
Volume: 100 L
Temperature: 15 ºC
2013 Pinot noir
Control
0 mg/L
Grape seed tannins (20 g/hL)
20 mg/L
0 mg/L
10 mg/L
Oxygen supply per month for 12 weeks
10 mg/L
Each experiment was carried out in duplicate.
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 15
Anthocyanins during micro-ox:
Impact of different oxygen rates
End12 wk micro-ox
120
Control
10 mg/L/month
20 mg/L/month
110
Nathocyanins (mg/L)
100
90
80
70
60
50
40
30
0
1
2
3
4
5
ASVO Seminar Inputs to Outputs; is less, more?
6
7
8
9
Time (weeks)
10
11
12
13
November 18th, 2014
14
15
16
Slide 16
Colour during Micro-ox:
Impact of different oxygen rates
End12 wk micro-ox
7
Control
10 mg/L/month
Color Density (AU)
20 mg/L/month
6
5
4
0
1
2
3
4
5
ASVO Seminar Inputs to Outputs; is less, more?
6
7
8
9
Time (weeks)
10
11
12
13
November 18th, 2014
14
15
16
Slide 17
Acetaldehyde during Micro-ox:
Impact of different oxygen rates
4 wk after Mox:
End12 wk micro-ox  35 ppm free SO
7
2
100
Control
10 mg/L/month
80
Total SO2: 109 mg/L (a)
6
60
Total SO2: 81 mg/L (b)
40
5
20
Total SO2: 84 mg/L (b)
4
0
0
1
2
3
4
5
ASVO Seminar Inputs to Outputs; is less, more?
6
7
8
9
Time (weeks)
10
11
12
13
November 18th, 2014
14
15
16
Slide 18
Acetaldehyde (mg/L)
Color Density (AU)
20 mg/L/month
Colour during Micro-ox:
Impact of tannins and different oxygen rates
End12 wk micro-ox
7
Control
10 mg/L/month
Tannin
Color Density (AU)
Tannin + 10 mg/L/month
6
Tannin effect
with Mox
5
Tannin effect
w/o O2
4
0
1
2
3
4
5
ASVO Seminar Inputs to Outputs; is less, more?
6
7
8
9
Time (weeks)
10
11
12
13
November 18th, 2014
14
15
16
Slide 19
Acetaldehyde during Micro-ox:
Impact of tannins and different oxygen rates
4 wk after Mox:
End12 wk micro-ox  35 ppm free SO
7
2
100
Control
10 mg/L/month
Tannin
Color Density (AU)
6
60
Total SO2: 86 mg/L (a)
Total SO2: 81 mg/L (a)
40
5
20
Total SO2: 79 mg/L (a)
Total SO2: 84 mg/L (a)
4
0
0
1
2
3
4
5
ASVO Seminar Inputs to Outputs; is less, more?
6
7
8
9
Time (weeks)
10
11
12
13
November 18th, 2014
14
15
16
Slide 20
Acetaldehyde (mg/L)
80
Tannin + 10 mg/L/month
Vitisin B and anthocyanin-catechin
oligomers after micro-ox
30
Trimer
25
Dimer
Concentration (mg/L)
Vitisin B
20
15
10
5
0
Control
10 mg/L/month
ASVO Seminar Inputs to Outputs; is less, more?
20 mg/L/month
Tannin
Tannin
+ 10 mg/L/month
November 18th, 2014
Slide 21
Color properties of malvidin vs.
anthocyanin-catechin oligomers
Absorption at 520 nm
Absorption at 620 nm
(1 g/L pure substance in model wine)
(1 g/L pure substance in model wine)
6
5
4
0.8
Absorption
Absorption
1.0
Absorption
Abs. after SO2
3
2
pH 1
pH 3.5
pH 5
0.6
0.4
0.2
1
0.0
0
Monomer
Dimer
Trimer
Molar absorptivity at 520 nm
Monomer
Dimer
Trimer
Ethylidene bridges:
Batochromic shift
at low pH
εpH1= 3892 L mol-1 cm-1
εpH1= 3137 L mol-1 cm-1
εpH1= 2200 L mol-1 cm-1
ASVO Seminar Inputs to Outputs; is less, more?
Weber et al. (2014) Food Res Int (in press)
November 18th, 2014
Slide 22
Sensory impact of tannins and Micro-ox in
2013 Pinot noir 3 month after bottling
4
10 mg O2/L/month
Control
Control
PC 2 (32 %)
2
Descriptive analysis
in June 2014;
n = 18 × 2; Scale: 0-10;
Significance levels:
* p ≤ 0,05
** p ≤ 0,01;
*** p ≤ 0,001
20 mg O2/L/month
0
-2
10 mg O2/L/month
No tannin addition
20 g/hL tannins
-4
-4
-2
ASVO Seminar Inputs to Outputs; is less, more?
0
PC 1 (49 %)
2
4
November 18th, 2014
Slide 23
Proportion of anthocyanins in polymeric pigments (mg/g)
Incorporated anthocyanins in polym. phenolic
fractions in a micro-oxed 2005 Cab. Sauv.
100
Descending molecular weight 
80
60
40
20
0
14 polym. phenolic fractions separated by Sephadex LH20
Weber et al. (2013) Am J Enol Vitic 64: 15
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 24
Astringeny of polym. phenolic fractions
in a micro-oxed 2005 Cab. Sauv.
10
Descending molecular weight 
Astringency intensity
8
6
Descriptive analysis
Iso-concentrations in water
n = 13 × 2; Scale: 0-10;
Values sharing the same letter
are not significantly different at
p < 0.05.
a
bcd bc
cde
4
fg
efg defg
efg
ab
a
bcd
cdef cdef
g
2
0
14 polym. phenolic fractions separated by Sephadex LH20
Weber et al. (2013) Am J Enol Vitic 64: 15
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 25
Conclusions
• Micro-ox only if laccase-free [and microbial safe]
• Micro-ox as early as possible: After AF (not during MLF)
• For Pinot some 5-10 mg O2/L/month after MLF for 3
months help for colour stabilisation and lower green
tannin perception
[Pre-MLF equivalent: 20-30 mg O2/L/month for 4 weeks]
• Slight accumulation of acetaldehyde is OK, as long as it
doesn’t cause a higher SO2 demand
• A good proportion of tannins is needed for the formation
of polymeric pigments
• Polymeric pigments does not only seem to improve
colour, but also to soften astringency perception
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 26
Thanks to
Patrick Nickolaus, Hai-Linh Trieu, Fabian Weber
Julieta Luna Zullo, Annie-Michelle Nno-Essaa,
Julia Willeke, Uli Fischer, Hans-Georg Schmarr,
Anette Schormann, Sascha Wolz,
Peter Winterhalter, Eva Schmalfuss
Industry partners for co-funding, providing equipment,
and enological supplements: Nomacorc, Ever Intec,
Lallemand, WZG
This research project is supported by the German Ministry
of Economics and Technology (via AiF) and the Research
Association of the German Food Industry (FEI) in
cooperation with the Association of German Wine-growers
(DWV) and the Association of Cooperative Wineries Baden
Württemberg (BWGV). Project AiF-FV 17719 N
ASVO Seminar Inputs to Outputs; is less, more?
November 18th, 2014
Slide 27