Early vs. late leaf removal strategies for ‘Pinot Noir’ (Vitis vinifera L.): effect on colour-related phenolics in young wines following alcoholic fermentation
Authors & Addresses Melita STERNAD LEMUT1, Kajetan TROST1, Paolo SIVILOTTI1, Panagiotis ARAPITSAS2, Urska VRHOVSEK2 *
1 2
University of Nova Gorica, Wine Research Centre, Glavni trg 8, SI-5271, Vipava, Slovenia;
Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach (FEM), via E. Mach 1, 38010 San Michele all'Adige, Italy
*
Corresponding author: Urska VRHOVSEK; e-mail:
[email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6193
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Abstract
BACKGROUND: Widely adopted viticultural practice of late (véraison) leaf removal is
now losing many of its advantages given nowadays-warmer vineyard conditions. With the scope of seeking a good alternative, the influence of earlier leaf removals (at pre-flowering and berry-set) on colour-related phenolics in young Pinot Noir wines was investigated in years 2009 and 2010.
RESULTS: Total flavonols in 2009 wines were 71% and 52% higher in case of véraison
and berry-set treatments respectively as compared to untreated controls, while in 2010 the average content of flavonols was highest with pre-flowering leaf removal (75% higher to control). Anthocyanin content in 2009 wines was 18% and 11% higher for véraison and berry-set respectively and favoured by early leaf removals in 2010 (50% and 43% higher in berry-set and pre-flowering respectively) as compared to control. Changes in hydroxycinnamic acid profiles were shown to be greatest in 2010 wines resulting from early leaf removal treatments. Promoted formation of vitisin A-like pigments in 2010 leaf removal treatments was observed during fermentation.
CONCLUSIONS: Phenolic profiles of grapes/wines were affected by leaf removal timing,
although differently in two (extremely different) seasons. Earlier strategies showed some promising results with good proportions mainly of flavonols and anthocyanins, kept also in young wines. Vitisins A in wines were positively affected by all leaf removals.
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Keywords: ‘Pinot Noir’; global warming; pre-flowering leaf removal; berry-set leaf removal; phenolic profiles; pyranoanthocyanins.
Introduction
The profiles of the main technologically important phenolic compounds in grapes are not only genotype dependent but can also be significantly affected by manipulation of grapevine environmental conditions.
1-3
Although winegrowers obviously cannot influence
the vintage/region macroclimate or site/vineyard mesoclimate conditions, it is in their power to significantly improve microclimate conditions within cluster area by carefully selecting viticulture techniques and moreover choosing the timing for their implementation at different grape development stages. Leaf removal (defoliation) is known to be one of the viticultural practices enabling successful canopy microclimate manipulation, leading to some important grape quality improvements,
1,4,5
although earlier application (at pre-flowering and berry-set) has been
less studied and there is still little information on their influence on wines from different grapevine varieties. 6 Within the context of global warming, many winegrowers are already facing the problem of a loss of effectiveness of widely adopted late (véraison) leaf removal, due to higher temperatures and severe UV exposure after canopy opening, leading to harmful sunburns on the grape berry skin tissue 7 as well as inhibition of the biosynthesis of some important phenolic compounds (e.g. anthocyanins).
8
On the other hand, earlier leaf
removal alternatives need to be well explored on the “from grapes to wine” basis before adapting them to any grapevine variety can be justified.
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Any significant vineyard management-related improvements in grape composition would normally be aimed to reflect also in the wines 9 but it is known that they can significantly or even totally lose their importance during the winemaking processes. The initial quantitative and qualitative input of flavonols, anthocyanins and hydroxycinnamic acids is probably most relevant for the development of red wine colour characteristics, which are particularly important for the ‘Pinot Noir’ (Vitis vinifera L.) grapevine variety, known for its poor (genetically derived) anthocyanin potential. Thus, any improvement in anthocyanin quantitative and/or qualitative profiles may be of considerable importance for ‘Pinot Noir’, even in terms of the simple anthocyanin glucosides normally found in relatively large quantities in other young red wines.
9
Better anthocyanin yield, combined with
improvements in other colour-related phenolics in young wines can offer greater potential for further formation of complex and more stable pigments such as pyranoanthocynanins and other related pigments, to the literature,
14
10-13
thus improving wine colour stability over time. According
there are two crucial factors for the formation of pyranoanthocyanins:
anthocyanins (with a minimum concentration corresponding to 18.5 mg L-1 as malvidin-3glucoside
15
) and their reaction partners (such as hydroxycinnamic acids, vinylphenols,
acetone and some further enolisable molecules). Apart from several oenological variables affecting the final yield of phenolics in wine during maceration/fermentation, their location in grape berry tissue and the chemical interactions (with each other and with other grape berry compounds) are known to limit the results in wine. Furthermore, some phenolic compounds are reported to be absorbed by the yeasts during fermentation, although with different intensities in individual compounds.
16
However, very little is known about the direct and/or indirect influence of early defoliation on reaction dynamics/chemical formation during cold maceration and alcoholic This article is protected by copyright. All rights reserved.
fermentation and finally the yield of colour-related phenolics in young Pinot Noir wines. This experiment was thus designed to reveal the reflection of vineyard microclimate modification efforts on the fate of related phenolics during typical Pinot Noir vinification processes.
Material and methods EXPERIMENTAL VINEYARD AND PLANT MATERIAL
The experiment was carried out in two subsequent years (2009 and 2010), observing 320 (240 in 2009) ‘Pinot Noir’ (V. vinifera L.) grapevines in total, located in the Vipava Valley (Slovenia) at the Potoce vineyard. The vineyard was planted in 2004 and it is Guyot trained, with a plant density of 5682 plants/he (0.8 m x 2.2 m), altitude of 95 m a.s.l. and is E-W row orientated. A completely randomised experimental design was set up with 16 plots of 5 vines per each treatment. Leaf removal was performed manually, removing the basal 4-6 leaves from all shoots: in 2009 defoliation was performed at berry-set (BS) (phenological stage BBCH 71) and at véraison (VE) (BBCH 83), while one third of the experimental vines were left untreated (UN) and used as a control. In 2010 a new and innovative viticulture technique of pre-flowering (PF) leaf removal (BBCH 57) was added to the observation and compared to the berry-set, véraison and control treatments.
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MUST FERMENTATION AND SAMPLING
Grapes from all the treatments were collected (in biological triplicates) separately at optimum maturity (based on the maturity level registered on UN grapes: 22 Brix and 5.5 g L-1 titratable acidity on average), reached on September 6 and September 20 in 2009 and 2010 respectively. All the grape samples were first de-stemmed, crushed, protected with 0.1g kg-1 K2S2O5 and mixed, before 20 litres of the pomace was separated and placed in 25L stainless steel experimental tanks (in total 3 tanks x 20L of pomace for each treatment, corresponding to biological triplicates collected from the vineyard). Experimental cold maceration and alcoholic fermentation were performed in the temperature-controlled chambers of the Wine Research Cellar, University of Nova Gorica. Each 20L batch was initially cooled down to 5°C and then subjected to typical ‘Pinot Noir’ winemaking treatments, starting with 48 hours of cold maceration (in a chamber set to 5°C) and followed by induced alcoholic fermentation once the temperature of the pomace had been brought back to room temperature (in a chamber set to 20°C). The yeast strain Saccharomyces cerevisiae “Fermol Premier Cru” type (AEB Group, Italy) was used to start fermentation (0.2 g L-1) and the yeast nutrient Fermoplus Starter (AEB Group, Italy) was added (0.3 g L-1) to achieve better continuation of the fermentation processes. Skin caps were pushed down and mixed every 12 hours, while the fermentation curve data (temperature, sugar level/density) were checked every 24 hours. The samples were collected: after cold maceration (CM); every 48 hours during alcoholic fermentation (after mixing the fermenting juice and skin caps); and finally after pressing in a pressurecontrolled experimental (30 L) mechanical press (Skrlj d.o.o., Slovenia), following the protocol of four (4 x 5 minutes) cycles (0.4/0.6/0.8/1.0 bar respectively). 20 mL of each This article is protected by copyright. All rights reserved.
must/wine under observation was added to 80 mL of methanol (hereafter MeOH extracts) in a dark glass container and stored at -20°C until the analysis was performed.
CLIMATE/MICROCLIMATE (BERRY SURFACE TEMPERATURE) MONITORING
The temperature on berry surfaces from vines receiving the different treatments was measured every two hours during three typical hot (August) days for both vintages under observation. 25 measurements per treatment were done at each observation point/time, using a Voltcraft IR-360 IR Thermom infrared thermometer (Conrad Electronics, Germany). Data on site climate conditions were collected with the help of an IMT300 iMetos meteorological station (Pessl Instruments, Germany), located in the experimental vineyard. Furthermore, data from the National Meteorological Service of Slovenia (EARSmeteo.si) were used to evaluate climatic conditions.
ANALYSIS OF GRAPE SAMPLES
In the case of grape samples, MeOH extracts of berry skins were first prepared
17
and the
analysis was then carried out according to already published protocols. 3 The separation and quantification of individual anthocyanins and flavonols was done using a Waters chromatographic system (Waters, MA, USA), made up of two 510 pumps, auto-sampler 717+ and UV-Vis dual wavelength 2487 detector. The total amounts were calculated as a sum of the individual compounds detected. All the analyses were performed in technical duplicates.
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ANALYSIS OF MUST/WINE SAMPLES
HPLC-DAD determination of flavonols: Methanol was first removed from the MeOH extracts using a rotary evaporator (Buechi, Switzerland). Individual flavonols in musts/wines were then analysed after flavonol glycosides had been subjected to acid hydrolysis, as previously described.
17
Further separation and quantification of flavonol
aglicons was performed using an Alliance 2695 HPLC instrument (Waters, MA, USA), equipped with DAD Waters 2996 (Waters, MA, USA). Total flavonols were calculated as the sum of the individual flavonols detected.
HPLC-VIS analysis of anthocyanins: The analytical method previously described
3
was
used to detect anthocyanins in the MeOH extracts of the musts and wines under observation. The separation and quantification of individual anthocyanins was performed using a Waters chromatographic system (Waters, MA, USA) made up of two 510 pumps, auto-sampler 717+ and UV–Vis dual wavelength 2487 detector. Total anthocyanins were calculated as the sum of the individual anthocyanins detected. All the analyses were performed in technical duplicates.
UHPLC-TQ-MS
determination
of
pyranoanthocyanins:
The
occurrence
of
pyranoanthocyanins was monitored using an ACQUITY Ultra Performance Liquid Chromatographic System (Waters, MA, USA), coupled to a Xevo TQ MS System (Waters, UK) and following published analytical procedure.
13
Sample preparation for the analysis
was previously carried out in accordance with reported protocol, 18 although a 20:80 MeOH extract was used instead of wine, and dilution with H2O was avoided. This article is protected by copyright. All rights reserved.
HPLC-DAD determination of hydroxycinnamic acids: The esters of hydroxycinnamic acids were determined based on the method previously presented by
19
with a minor
adjustment of sample preparation introduced to existing sample material. An aliquot of 10 mL of MeOH extract was first evaporated to dryness using a solvent evaporator (EZ-2, GeneVac Ltd., UK) under reduced pressure at 45ºC. The sample was then reconstructed in a quantitative flask up to 1mL of the final volume with 0.5% formic acid and filtered through 0.45 μm, 13 mm PTFE syringe-tip filters (Millipore, Bedford, USA) into vials. The separation and quantification of hydroxycinnamates was then performed on a HPLC instrument Alliance 2695 (Waters, MA, USA), equipped with DAD Waters 2996 (Waters, MA, USA).
UV-VIS spectrophotometric determination of colour properties: Colour measurements were done with the help of a HP UV/VIS Spektrofotometer Lambda 35 (PerkinElmer, UK), using the procedure as previously described.
20
The CIE-LAB parameters L * a * and b *
were determined following the recommendations of the Commission Internationale de L’Eclariage. 21
STATISTICAL ANALYSIS
Grape, must and wine composition data were statistically analysed using one-way ANOVA to test the significance of treatments at harvest (grapes) and at different stages of cold maceration/alcoholic fermentation (musts/wines) (P
