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EMaVE Consortium - Vinifera Euromaster

European Master of Science (MSc) in Viticulture and Enology

UNIVERSITA’DEGLI STUDI DI UDINE

Laurea Magistrale in Viticoltura, Enologia e Mercati vitivinicoli

“Radical scavenging activity and oxygen consumption of different

oenological products and additives ”

MASTER THESIS developed in the

Food Science Department of the University of Udine

Presented by Mariana Silvina Páez

Vinifera Euromaster 2010-2012

Main Supervisor: Professore Roberto Zironi

Co- Supervisor: Dottore Piergiorgio Comuzzo

Academic Year 2010-2012

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ABSTRACT

The use of sulfur dioxide in winemaking seems indispensable for its properties of

antiseptic, antioxidasic and antioxidant.

Other molecules and additives have been proposed for replacing it in winemaking,

but poor investigations on their effectiveness are available from the technological

point of view.

For this reason, in this study, some oenological additives and products (ascorbic

acid, glutathione, yeast lees, and an inactive dry yeast preparation) have been

compared with sulfur dioxide for their radical scavenging activity (DPPH assay)

and oxygen consumption capacity. Experiments were carried out in wine- like

solution and in wine.

Concerning DPPH assay, results demonstrated that some of the tested products

showed good performances as radical-scavengers, compared to sulfites; but

others, like ascorbic acid, behaved very similar to SO2. In addition, yeast lees and

extracts (IDY) showed the highest radical scavenging activity and huge sulfite

concentrations (500 mg/L) were needed to give similar effects.

Glutathione ability to discolor DPPH, in comparison with sulfur dioxide, was

different in model solution and in the different wines tested, so that the anti-

radical action of both these additives in wine could seem also connected with wine

characteristics and composition.

Different wines had different oxygen consumption curves and the effect of the

tested treatments was differently evident in the two tested wines, being less clear

in the youngest one. Yeast lees generally demonstrated a higher ability to promote

oxygen consumption; their action was similar to that of ascorbic acid. Contrarily,

inactive dry yeasts (IDY), gave high oxygen consumption at the beginning of the

monitoring time, reducing the consumption rate after some days; at the end of the

monitoring period (two weeks) the wines treated with IDY showed generally the

highest oxygen concentration. IDY could sometimes give positive effects on wine

color protection.

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Index

1.INTRODUCTION ....................................................................................... ¡ERROR! MARCADOR NO DEFINIDO.

1.1. MECHANISMS OF OXIDATION IN WINE ..................................................................................................... 6

1.1.1. Enzymatic oxidation ....................................................................................................................... 10

1.1.2.Non- enzymatic oxidation ................................................................................................................... 11

1.2. SUBSTANCES USED IN VINIFICATION AS ANTIOXIDANTS ............................................................................... 12

1.2.1. Sulphur dioxide .............................................................................................................................. 12

1.2.2. Ascorbic acid .................................................................................................................................. 14

1.2.3. Glutathione .................................................................................................................................... 15

1.2.4. Yeast lees and Yeast derivatives (inactive dry yeast) ..................................................................... 18

1.2.5. Combined effect of wine antioxidants ........................................................................................... 20

1.3. AIM OF THIS WORK ............................................................................................................................ 22

2. MATERIALS AND METHODS ................................................................................................................... 23

2.1. CHEMICALS AND OENOLOGICAL PRODUCTS .............................................................................................. 23

2.2. EVALUATION OF THE RADICAL SCAVENGING ACTIVITY OF DIFFERENT OENOLOGICAL ADDITIVES AND PRODUCTS ......... 23

2.2.1.Samples preparation ........................................................................................................................... 23

2.2.2. DPPH assay .................................................................................................................................... 24

2.3. EFFECT OF DIFFERENT OENOLOGICAL ADDITIVES AND PRODUCTS ON WINE OXYGEN CONSUMPTION ....................... 24

2.3.1. Samples preparation ...................................................................................................................... 24

2.3.3. Spectrophotometric measurements .............................................................................................. 27

2.4. STATISTICAL ANALYSIS ........................................................................................................................ 28

3. RESULTS AND DISCUSSION .................................................................................................................... 29

3.1. STUDY OF THE BEHAVIOR OF SOME ANTIOXIDANT AGENTS IN WINE-LIKE BUFFER AND IN WHITE WINE USING DPPH

STABLE FREE RADICAL. ...................................................................................................................................... 29

3.1.2. Results of DPPH trials in white wine .............................................................................................. 30

3.1.3. Results of DPPH trials in white wine and the antioxidant agents in pairs ..................................... 32

3.2. EFFECT OF DIFFERENT OENOLOGICAL ADDITIVES AND PRODUCTS ON WINE OXYGEN CONSUMPTION CAPACITY .......... 34

4. CONCLUSIONS ....................................................................................................................................... 45

5. BIBLIOGRAPHY ....................................................................................................................................... 46

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List of figures and tables

Figure 1: Most common flavonoid compounds in wine. .......................................................... 7

Figure 2: Most common Non-flavonoid compounds in wine. ............................................... 8

Figure 3: Oxygen reduction ................................................................................................................. 9

Figure 4: Enzymatic browning process in grape must. ........................................................... 10

Figure 5: Fenton reaction .................................................................................................................. 12

Figure 6: Reduction of copper by ascorbic acid and next oxidation by hydroxy

peroxide .................................................................................................................................................... 15

Figure 7: Glutathione .......................................................................................................................... 17

Figure 8: Glutathione oxidation ...................................................................................................... 17

Figure 9: OxySense® equipment to measure the oxygen consumption. ....................... 27

Figure 10: POM -test calculation .................................................................................................... 27

Figure 11: DPPH-test. Radical scavenging activity of different enological additives

and products, in wine-like solution ................................................................................................ 29

Figure 12: DPPH- test. Radical scavenging activity of a table wine treated with

different enological additives and products ............................................................................... 31

Figure 13: DPPH test. Radical scavenging activity of different enological additives

and products isolated and in pairs in a table white wine ....................................................... 33

Figure 14: Oxygen consumption curves of a white wine (Friulano, D.O.C. Colli

Orientali del Friuli- Italy, vintage 2007) treated with different enological additives

and products. .......................................................................................................................................... 35

Figure 15: One way ANOVA, carried out on the slopes of the lines obtained by

linearization (log 10) of the curves in figure 14. .......................................................................... 36

Figure 16: Oxygen consumption curves registered in a white wine ( Chardonnay,

D.O.C. Grave del Friuli- Italy, vintage 2011) treated with different enological

additives and products. ....................................................................................................................... 40

Figure 17: One way ANOVA, carried out on the slopes of the lines obtained by

transformation ( log 10) of the curves in Figure 16. ................................................................. 41

Table 1: Amounts of enological additives and products added in the two sets of

trials ............................................................................................................................................................ 25

Table 2: Equations of the trend lines obtained by transforming (log 10) the curves

of oxygen consumption as detected for the different Friulano wine samples

analyzed in Figure 14 (average curves) and Figure 15 (ANOVA on slopes). Slopes

and R2 values are reported. ............................................................................................................... 37

Table 3: Oxygen concentration, DO 420nm and POM test detected for the Friulano

wine at the end of the measures ..................................................................................................... 38

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Table 4: Equations of the trend lines obtained by transforming (log 10) the curves

in Figure 16 (average curves) and figure 17 (ANOVA on slopes). Slopes and R2

values are reported ............................................................................................................................... 42

Table 5: Oxygen concentration, DO 420nm and POM test detected for the wines at

the end of the measures ..................................................................................................................... 43

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1. INTRODUCTION

1.1. Mechanisms of oxidation in wine

Oxidative browning is a long-standing problem in winemaking.

Wine oxidation can be divided in enzymatic oxidation and non-enzymatic

oxidation. Enzymatic oxidation almost entirely occurs in grape must and is largely

correlated with the content of phenols like the hydroxycinnamates, such as

caffeoyltartaric acid, paracoumaroyltartaric acid, and flavan-3-ols. Non-enzymatic

oxidation, also called chemical oxidation, prevails in wine and begin by the

oxidation of polyphenols containing a catechol or a galloyl group, in presence of

catalysts (e.g. iron, or copper). These phenolic reactions, both enzymatic and non-

enzymatic, result in by-products named quinones, (C.M. Oliveira et al., 2011).

Constituents of both red and white wines are capable of reacting with significant

amounts of oxygen, polyphenols being among the most readily oxidized wine

constituents.

Phenolic substances

Wine polyphenolic substances are usually subdivided into two groups: flavonoids

and Non-flavonoid compounds, (Figure 1, and Figure 2).

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Figure 1: Most common flavonoid compounds in wine.

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Figure 2: Most common Non-flavonoid compounds in wine.

The concentration of flavonoids in wine is strongly affected by the winemaking

practices and the degree of extraction from skins and seeds. Flavan-3-ols are

found in the solid parts of the berry (seed, skin and stem) in monomeric,

oligomeric, or polymeric forms. The latter two forms are also known as

proanthocyanidins or condensed tannins. While seed tannins are oligomers and

polymers composed of the monomeric flavan-3-ols (+)-catechin, (−) epicatechin,

and (−)-epicatechin gallate (Prieur, C et al., 1994), skin tannins also contain (−)-

epigallocatechin and trace amounts of (+)- gallocatechin and (−)-epigallocatechin

gallate (Escribano-Bailón, M. T.et al., 1995); (Souquet, J. M. et al. , 1996) .Flavan-3-

ols have been reported to exhibit several health beneficial effects by acting as

antioxidant, anticarcinogen, cardiopreventive, antimicrobial, anti-viral, and neuro-

protective agents ( P.M.Aron and J.A.Kennedy, 2007). The seeds contain higher

concentrations of monomeric, oligomeric, and polymeric flavan-3-ols than the

skins. Levels of proanthocyanidins or condensed tannins are between 1 g/L and 4

g/L in red wines while in white wine, levels are in the range of 100 mg/L and highly

dependent on pressing techniques, (Ribéreau-Gayon, P., Glories, Y., Maugean, A., &

Dubourdieu, D. ,2000).

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Non-flavonoid compounds are mainly derivatives of benzoic acid and of cinnamic

acid. Another class of non-flavonoids in grape includes the stilbenes and stilbene

glycosides, trans resveratrol being the best known example (Figure 2). A different

class of nonflavonoids in wines are the hydrolysable tannins that are derived from

oak. (C.M. Oliveira et al., 2011)

Red wines contain polyphenols at a higher concentration than white wines

(Castellari, M.et al., 2000). White wines containing mainly hydroxycinnamic acids,

but these remain very important for oxidation related issues in wine browning and

losses of varietal aroma. (Betes-Saura, C. et al., 1996)

Reactive oxygen species

Reactive oxygen species (ROS) is a collective term used to describe oxygen

radicals, such as superoxide anion (O2•−) and its conjugate acid hydroperoxyl

(HOO•), hydroxyl (HO•), peroxyl (ROO•), alkoxyl (RO•) radicals, and certain other

non-radicals that are either potential oxidizing agents or are easily converted into

radicals, such as hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid (HOCl),

singlet oxygen (1O2), and lipid peroxides (LOOH),(Pourova, J. et al., 2010).

In wine, ROS can be produced by reduced transition metals ions [e.g. Fe(II)]. The

initial transfer of an electron leads to the formation of superoxide radical anion

(O2 •−), which at wine pH exists in the protonated form hydroperoxyl radical

(HOO•) .The transfer of a second electron will produce peroxide anion (O22−), which

at wine pH exists in the protonated form hydrogen peroxide (H2O2) . The next

reduction step creates an even more reactive oxidant, the hydroxyl radical (HO•),

which can abstract a hydrogen atom from organic compounds to produce water,

the final oxygen reduction product, (Figure 3), (Danilewicz, J. C., 2003);

(Waterhouse, A. L., & Laurie, V. F, 2006).

Figure 3: Oxygen reduction (Waterhouse & Laurie, 2006)

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1.1.1. Enzymatic oxidation

The enzymatic oxidation occurs almost entirely in grape must. A likely mechanism

for oxidation of phenolic compounds involves hydroxylation to the ortho-position

adjacent to an existing hydroxyl group of the phenolic substrate (monophenol

oxidase activity), and oxidation of ortho-dihydroxybenzenes to ortho-

benzoquinones (diphenol oxidase activity). Several classes of enzymes,

oxidoreductases, can catalyze these reactions. This class of enzymes includes the

well known Polyphenoloxidase, Peroxidase and laccase. (C.M. Oliveira et al., 2011)

In grape must, enzymatic browning (Figure 4) is largely correlated with the

content of hydroxycinnamates such as caffeoyltartaric acid (caftaric acid) and para-

coumaroyltartaric acid (coutaric acid), ( Cheynier, V. F.et al., 1986.), and is promoted

by flavan-3-ols . When grapes are crushed, polyphenoloxidases (PPO) are released,

and rapidly oxidize the hydroxycinnamates to benzoquinones. Meanwhile, the

benzoquinones produced by enzymatic oxidation will undergo further reactions,

according to their redox properties and electronic affinities.(Robards, K., et al.

,1999.). Being oxidants, quinones, can oxidize substances which have a lower

potential such as polyphenols and ascorbic acid as well SO2. The quinone is then

reduced back to its original catechol. (Kutyrev & Moskva, 1991).

Figure 4: Enzymatic browning process in grape must. (Li, H., Guo, A., & Wang, H.,

2008)

White must hyperoxygenation decreases the browning potential of wine in two

ways: by the tyrosinase disappearance and by the depletion of oxidable

polyphenols during the oxidation reactions. These results in wines with low

polyphenol concentrations and high GRP (Grape reaction Product) content (see in

the section 1.2.3. Glutathione oxidation), which are more stable than those made

from non-oxidized juice, in which high polyphenols contents are maintained with a

high browning potential (Li, H., Guo, A., & Wang, H. ,2008).

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1.1.2. Non- enzymatic oxidation

Non-enzymatic oxidation, also called chemical oxidation, prevails in wine with the

characteristics of regeneration and autocatalysis, and it may also occur through the

direct reaction with light (Main, G. L., 1992). O-Diphenols, mainly including caffeic

acid and its esters, catechin, epicatechin, anthocyanins and their derivatives, and

gallic acid, are considered to be the most susceptible to oxidation in non-

enzymatic browning process, and the levels of flavan 3-ols are most significantly

correlated to the browning degree of most white wines (Fernández-Zurbano, P.,

Ferreira, V., Escudero, A., & Cacho, J., 1998);(Fernández-Zurbano, P. et al., 1995);

(Lopez-Toledano, A et al., 2002); (Saucier, C. T., & Waterhouse, A. L., 1999)

Non-enzymatic browning in wine can arise through several pathways related to

phenols, and one of them is the oxidation of phenols and subsequent

polymerization of the oxidized products, the first process catalyzed by copper and

iron. Other routes involve polymerization reactions between phenols and other

compounds present in wine, including condensation with acetaldehyde or glyoxylic

acid (derived from the oxidation of tartaric acid). The oxidation of ethanol

catalyzed by transition metals or through coupled oxidation of phenols produce

some acetaldehyde in wine (Sullivan, P. J.,2002); (Wildenradt, H. L.,&Singleton, V. L.,

1974).

It had been examinated a mechanism by which oxygen and its intermediate

reducing products react with wine constituents, as well the participation of

transition metal ions in these reactions with the conclusion that oxygen does not

react directly with phenolic compounds without the presence of transition metal

ions, (Waterhouse, A. L., & Laurie, V. F, 2006).

During the process of non-enzymatic oxidation of wine, o-diphenols are oxidized

to o-quinones and semi-quinone, and free radicals may be produced, while oxygen

is reduced to H2O2, (Waterhouse, A. L., & Laurie, V. F, 2006).

The quinones formed during the process as the primary products are unstable and

may undergo further reactions. These reactions, which may cause pigment

formation, are similar to those taking place in enzymatic browning despite the

quinones from enzymatic or non-enzymatic oxidation (Robards, K. et al.,1999).;

(Singleton, V. L.,1987), for example, condensation reactions to form colored

products with high molecular weight or reduction reactions to generate original

phenols through trapping hydrogen atoms from other compounds.

Hydrogen peroxide (H2O2) may be formed during the oxidation of wine phenols,

which has been widely accepted.

Some authors also state that the main antioxidant function of SO2 in wine is to

react with H2O2, thus limiting the oxidation of ethanol and other saturated

hydroxyl compounds (Danilewicz, J. C., 2007). This should be taken into account,

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because H2O2 in association with ferrous ion (Fe2+) tends to generate reactive

oxygen species such as hydroxyl radical ,(HO•), which is known as the Fenton

reaction (Figure 5) (Choe, E., &Min, D. B. ,2005); (Zhao, B., 1999); (Waterhouse, A.

L., & Laurie, V. F, 2006).

Figure 5: Fenton reaction

Hydroxyl radical, (HO•), a reduced product of oxygen with short existing life of

about 10-6 s in water, is presently the most powerful oxidant in reactive oxygen

species and capable of rapidly oxidizing most organic substances (Choe, E., &Min,

D. B., 2005). Some authors suggest that (HO•) is able to oxidize almost any

component found in wine, nearly in proportion to their concentrations (Laurie &

Waterhouse 2006). However, (HO•), is non-selective and only reacts with adjacent

molecules on account of its smaller action radius (Zhao, B., 1999); (Waterhouse, A.

L., & Laurie, V. F, 2006).

Thus, ethanol and tartaric acid being the prime substrates due to their relatively

large concentrations in wine would likely be oxidized by (HO•) to form

acetaldehyde and glyoxylic acid, respectively, and other abundant components of

wine such as glycerol, sugars and organic acids probably undergo the same kind of

oxidation reactions to yield corresponding products .These expected oxidation

products, mainly aldehydes and ketones, are good nucleophiles, which may be

important to colour development and other changes to tannin structure (Li, H.,

Guo, A., & Wang, H. ,2008);(Waterhouse, A. L., & Laurie, V. F, 2006).

1.2. Substances used in vinification as antioxidants

1.2.1. Sulphur dioxide

The use of sulfur dioxide in winemaking seems indispensable for its properties of

antiseptic, antioxidasic and antioxidant capacity. It has long been used in

winemaking to inhibit oxidation and growth of undesirable micro-organisms

including wild yeast, and acetic and lactic bacteria. Concentrations of added SO2

to wine generally range from 50 to 200 mg/L.

In wine, there is an equilibrium between molecular and ionic forms of sulfur

dioxide. At wine pH, 94 to 99% exists in the ionic form as the bisulfite ion HSO3-

and so only a small proportion is present as free SO2. Once in wine solution, sulfur

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dioxide can bind with several molecules such as acetaldehyde, anthocyanins,

pyruvic acid, ketoglutaric acid, glucose or phenolic compounds, particularly,

caffeic acid and p-coumaric acid. There are commonly two fractions of SO2 in wine:

the “free SO2”, referred to HSO3- and SO2, and the“bound SO2”, indicating

sulfur dioxide that is mainly bound to unsaturated compounds. Sulfur dioxide does

not react directly with oxygen but with the oxygen reduced form, hydrogen

peroxide (Elias, R. J., Andersen, & Waterhouse, A. L., 2010) .In this way, SO2 can

inhibit aldehydes formation by competing for hydrogen peroxide. Sulphur dioxide

also plays an important role in reducing quinones formed during oxidation process

back to their phenol form, (C.M. Oliveira et al., 2011).

However, as a result of potential health problems that may arise like some allergies

caused by SO2-derived compounds, namely the sulfites, causing symptoms such as

headaches, nausea, gastric irritation, and breathing difficulties in asthma patients.

Consequently, the legislated maximum concentration of SO2 allowed in wines has

been gradually reduced, (MC Santos et al., 2011).

Some wineries make wines in total or near-total absence of sulfur dioxide but it

would certainly be presumptuous to claim that all of the wines produced in the

various wineries throughout the world could be made in this manner. It must also

be taken into account that yeasts produce small quantities of SO2 during

fermentation. The commercial yeast normally used in winemaking, produce rarely

more than 10 mg/L but sometimes more than 30mg/L. Therefore we can not find a

wine without sulfur dioxide even in the absence of sulfiting, (P. Ribéreau-Gayon, D.

Dubourdieu, B. Donéche and A.Lonvaud, 2006). Other authors have found that

most yeast strains produce less than 30 mg/L total SO2, although some have been

reported to produce more than 100 mg/L (Osborne and Edwards, 2005

Eschenbruch, 1974; Weeks, 1969).

In Europe, wines must now prominently display on the bottle, next to restrictions

required by law, the presence of total sulfites in excess of 10 mg/L (European

Union Regulation 1991/2004).

In recent years as a result of potential health problems that may arise, the use of

sulphur dioxide in wine has recently come under review, (COMMISSION

REGULATION (EC) No 606/2009 of 10 July 2009, Annex IB). They have reduced in

10 mg/L the maximum total sulphur dioxide content in wines. For example, before

this law, for white wines the total sulphur dioxide content allowed was 210 mg/L

and now the maximum is 200 mg/L.

The use of sulphur dioxide in excessive doses must be avoided not only for health

reasons but also because, from an enological point of view, it can cause

organoleptic alterations in the final product, neutralize the aroma and even

produce characteristic aroma defects, (Ribéreau-Gayon P, Glories Y, Maujean A,

Dubourdieu D,2006).

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We can produce a wine reducing the total use of sulfur dioxide using some tools.

Winemakers are therefore faced with the problem of finding other preservatives

and innovative technologies, harmless to health, that can replace or at least

complement the action of SO2, making possible to reduce its levels in wines.

1.2.2. Ascorbic acid

Ascorbic acid is a natural compound which is found in most fruits and vegetables.

In grapes the level of ascorbic acid is generally found to vary between 5 to 150

mg/kg of fruit, (Zoecklein et al., 1995). The content after crushing and pressing

ranges from 10 to100 mg/L, (Rankine B., 2002).

Ascorbic acid has long been used in the wine industry as an antioxidant, the reason

being its ability to rapidly remove molecular oxygen from juice or wine. It is readily

oxidized under the conditions found in white wine. This is claimed as the basis of

its ability to protect other oxidizable wine constituents, including phenolic and

flavor compounds.

Due to the limited number of microbiological spoilage inhibitory functions of

ascorbic acid compared with those of sulfur dioxide, it was appreciated that

ascorbic acid should only be employed in a complementary role with sulfur dioxide

and not as a replacement. The combined use of ascorbic acid and sulfur dioxide

was initially seen to have many advantages over the use of either compound alone,

(Zoecklein et al., 1995); (Rankine B., 2002). These advantages included a reduction

in the level of required sulfur dioxide reducing the severe health problems allergy-

like reactions, (M. P. Bradshaw, et al., 2011)

The benefit of ascorbic acid as an antioxidant in white wine is its capacity to

scavenge molecular oxygen before the oxidation of phenolic compounds. In

comparison, sulfur dioxide is not nearly as efficient at scavenging molecular

oxygen (Danilewicz et al., 2008). Furthermore, if oxidation of phenolic compounds

does occur then ascorbic acid is also suggested to readily reduce the oxidized

products (i.e., ortho-quinones) back to the original phenolic state (Boulton et al.,

1996).

The“crossover”effect

It was demonstrated that ascorbic acid can exhibit a “crossover” effect

depending upon the level of the acid; (Buettner, G. R. and Jurkiewicz, B. A., 1996). It

was shown that at higher concentrations, ascorbic acid could act as an antioxidant,

while at lower concentrations a pro-oxidant influence could be demonstrated. The

authors, Buettner and Jurkiewicz, proposed that the crossover effect resulted from

the generation of radical species derived from the metal- catalized oxidation of

ascorbic acid.

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Ascorbic acid in the presence of metal ions and in the absence of sulfur dioxide

can actually acts as a free-radical initiator. There is evidence to suggest that under

certain conditions ascorbic acid may display pro-oxidant activity in the case where

sulfur dioxide is not present. The mechanism of this activity may be linked to the

presence of catalytic metals such as Fe3+ and Cu2+, which may be reduced by

ascorbic acid and enter redox cycling reactions.

For example, Moreaux et al. (1996), have shown that ascorbic acid may reduce Cu2+

to Cu+. (Figure 6)

AH2 + O2 + Cu2+

→ A + H2O2 + Cu+

H2O2 + Cu+ →• OH + OH

– + Cu

2+

Figure 6: Reduction of copper by ascorbic acid and next oxidation by hydroxy

peroxide

With the generated hydroxyl radicals and so, initiating damaging chain reactions.

Similarly Fe3+ may be reduced to Fe2+ by ascorbic acid. The difference between ascorbic acid acting as an antioxidant or pro-oxidant in the

absence of sulfur dioxide is then related to the relative concentrations of ascorbic

acid and the catalytically active metal ions, (M.P. Bradshaw et al., 2011). With high

concentrations of ascorbic acid, the chain length of the radical processes will be

small, due to its antioxidant effect. That is, radicals generated will be quenched by

the presence of ascorbic acid. However, as the concentration of ascorbic acid is

lowered, initiation processes will be slowed, but so too will antioxidant reactions.

Thus there will be a corresponding increase in the chain length of oxidatively-

damaging reactions. Hence under these conditions, ascorbate will have pro-

oxidant activity (M.P. Bradshaw et al., 2011), (Buettner, G. R. and Jurkiewicz, B. A.,

1996).

1.2.3. Glutathione

Glutathione, γ-L-Glutamyl-L-cysteinylglycine (GSH), is an important antioxidant

coming from the grapevine as it aids in decreasing aroma loss and the browning

that occurs due to oxidative processes in white wine.

In grapes, GSH concentration can exceed 100 mg/ kg according to grape cultivar,

environmental conditions and viticultural practices. The GSH content in grape juice

ranges from 10 to 100 mg /L (Cheynier, V., Souquet, J. M., & Moutounet, M., 1989)

and factors such as exposure to oxygen, tyrosinase activity and grape skin

maceration during pre-fermentation can affect its concentration (Du Toit, W. J.et

al.,2007); (Maggu, M.et al., 2007).

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The concentration of GSH in wine is lower than in juice and grapes and it ranges

from 1 to 20 mg/ L (Cassol, T., & Adams, D. O, 1995); (Du Toit, W. J.et al., 2007). In

instances where the concentration of GSH in white wine exceeds 6–10 mg/ L, both

colour and aroma are better preserved during ageing and storage (Lavigne, V., &

Dubourdieu, D.,2004). Saccharomyces cerevisiae can also affect the GSH content in

wine during alcoholic fermentation as well as during the ageing on the lees

(Lavigne, V., Pons, A., & Dudourdieu, D., 2007). Sonni, F. et al., 2011 mentioned

normal concentrations of Glutathione found in wine of 30 mg/L.

GSH plays a crucial role in the limitation of phenol oxidation during winemaking as

it can react with caftaric acid, generating 2-S-glutathionyl caftaric acid, also known

as Grape Reaction Product (GRP). In this way the formation of o-quinones and,

consequently, the production of browning polymers, is limited. GSH can thus have

a positive effect on white wine colour, appearing to make the color more stable

during ageing (Fracassetti D. et al., 2011).

GSH exerts a protective effect on certain wine aromas (Lavigne, V., & Dubourdieu,

D., 2004). It may lead to lower o-quinone-thiol associations, by competing for the

o-quinones, thereby leading to higher amounts of thiol-related aromas in wine.

GSH can also preserve aroma compounds, such as isoamyl acetate (3-methyl-1-

butyl acetate), ethyl hexanoate, and linalool (3, 7-dimethylocta-1, 6-dien- 3-ol)

during bottle storage (Papadopoulou, D., & Roussis, I. G., 2008). This aromatic

protection by GSH seems to be highly active in the presence of phenols, especially

when caffeic acid is dissolved in wine at certain levels (Roussis, I. G., Lambropoulos,

I., & Tzimas, P., 2007). It can also reduce the formation of sotolon (3-hydroxy-4,5-

dimethyl-2(5H)furanone), a compound responsible for the atypical ageing

character of white wine (Lavigne, V., & Dubourdieu, D., 2004).

Glutathione oxidation:

The thiol group (–SH) of the cystein in GSH acts as proton donor for unstable

molecules (reactive oxygen species, ROS, for example: H2O2), GSH assist in

preventing ROS damage by scavenging free radicals. GSH reduces disulfide bonds

and is converted to its oxidized form, Glutathione disulfide (GSSH), (Figure 7 &

Figure 8).

Once oxidized, glutathione can be reduced back by glutathione reductase, using

NADPH as an electron donor. The ratio of reduced glutathione to oxidized

glutathione is often used as a measure of oxidative stress. Generally glutathione is

found like GSH, if does exist GSSG in bigger quantity we can consider the

oxidation.

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Figure 7: Glutathione

H2O2 + 2GSH------- GSSG + 2 H2O.

Figure 8: Glutathione oxidation

GSH in the winemaking process:

GSH represents the 1% of the dry weight of S. cerevisiae, having in this way GSH in

the preparations of Inactive dry yeast (IDY). It had been proved that the content of

GSH depends of the yeast strain present in the wine. Therefore, the IDY

preparations could release different contents of GSH in the wine depending on the

yeast strain used in its industrial production having different effect on the wine

treated with them.

It had been proved that the addition of GSH (10 mg/L) before bottling of wine can

reduce the oxidation phenomena, maintaining the color and varietal aroma and

decreasing the formation of negative aroma compounds (Pozo-Bayón M.A. et al.,

2009).

The role of GSH in grape must oxidation:

In must oxidation, the initial oxygen uptake by o-dihydroxybenzenes is slowed by

addition of thiols like cysteine (Cys) or glutathione (GSH).When caftaric acid is

oxidized to its corresponding quinone by tyrosinase, GSH will quickly react with

the quinone forming a colorless product called grape reaction product (GRP; 2-S-

glutathionyl caftaric acid) which is no longer a substrate for further oxidation by

tyrosinase. Therefore, the formation of GRP is believed to limit the must browning

and depends on the relative amounts of GSH. Analysis of aged bottled wines

shows that GRP is slowly hydrolyzed to the GSH-caffeic acid derivative (the tartrate

ester is hydrolyzed). The specific brown products are not well characterized, but it

appears that the hydroxycinnamate quinones react with flavan-3-ols to form

colored products (C.M. Oliveira et al., 2011 ).

Page | 18

Sulphur dioxide (SO2) inhibits tyrosinase (Dubernet, M., & Ribereau-Gayon, P.,

1973) and prevents the production of GRP, which will maintain a high level of free

hydroxycinnamates with high browning potential. Moreover, unlike tyrosinase,

laccase will readily oxidize GRP. GRP was shown to be oxidized by laccase from

Botrytis cinerea to the corresponding o-quinone with substitution of the latter by

glutathione. When no glutathione is available, polymerization of the quinones led

to browning of the juice. It is accepted that tyrosinase is more sensitive to SO2,

while laccase is more resistant to SO2 and has a wider substrate oxidation

spectrum (Du Toit, W. J. et al., 2006).

1.2.4. Yeast lees and Yeast derivatives (inactive dry yeast)

Contact with yeast lees seems to protect wine from oxidation, contributing to the

prevention of browning (Caridi, A., 2006), (Pérez-Serradilla, J.A. & Luque de Castro,

M.D., 2008), (Palomero et al., 2009) and the development of oxidation-related

volatiles (Cullere et al., 2007).

Different accelerated oxidation tests showed a significantly lower degree of

oxidative alteration when assayed in the presence of yeasts lees. The prevention of

wine browning could be an indirect effect of the absorption of colored compounds

by lees but this resistance could also be ascribed to yeast-promoted protection.

The protective effect of lees could be largely due to the release of intracellular

compounds to the wine, as well as to membrane lipids, which consume oxygen

during wine aging, thus preventing wine oxidation (A.López-Toledano et al, 2006).

The cell wall makes up between 25 and 50% of cell volume (Lipke, P.N., Ovalle, R.,

1998) and consists of an inner three-dimensional network of ramified glucans and

outer layer of mannoproteins (Kath, F. and Kulicke, W.M., 1999), (Gemmill, T.R.and

Trimble, R.B., 1999). Yeast cells are enclosed by a cell wall containing 29–64% b-

glucans, 31% mannans, 13% proteins, 9% lipids and 1–2% chitin. The exact

structure and composition of the yeast cell wall depends strongly on the

cultivation conditions and with the mode of cell cultivation. It was found that the

dry mass and polysaccharides content of the cell wall could vary by more than 50%

with the nature of the carbon source, nitrogen limitation, pH, temperature and

aeration”, and with the mode of cell cultivation and strain (Aguilar-Uscanga, B., &

Francois, J. M., 2003).

The antioxidant activity of these wall biomolecules could also occur during sur lie

aging contributing to prevent oxidation, and these interactions deserve to be

clarified.

The studies on spent brewer's yeast have demonstrated that much of yeast wall

activity depends on the exposure of the reactive groups, such as protein aromatic

side chains and thiols, to substrate. The antioxidant activity of different cell wall

fractions of spent brewer's yeast (Saccharomyces cerevisiae), proved the significant

Page | 19

antioxidant activity for wall proteins and glucans (Jaehrig et al., 2007); (Jaehrig et

al,2008).

At enological aging conditions, i.e. without cell disruption and fraction isolation,

some of these biomolecules may hardly interact with the wine because they

constitute inner layers of the cell wall or because intra and intermolecular

interactions make the reactive groups inaccessible.

It had been found that (1-3)-b-D-glucan cell wall polysaccharide from the yeast

Saccharomyces cerevisiae exhibits antioxidative capabilities. In some studies, the

antioxidative activity of glucans from the cell walls of S. cerevisiae grown on

different media was investigated. The results show significant differences in the

glucan content of the cell walls and in their antioxidative activities depending on

growth medium. However, glucan itself seems to have a low antioxidative activity

in contrast to other cell wall fractions e.g. proteins (Jaehrig et al, 2008). Also it had

been found that carboxymethylated (1-3)-b-D-glucan from S. cerevisiae cell walls

exhibits antioxidative activity in terms of free radical scavenging (Kogan, G., Stasko,

A., Bauerova, K., Polovka, M., Soltes, L., Brezova,V., et al. (2005). Saccharomyces cerevisiae not only contains antioxidants in the cell wall but also in the cytoplasm

which holds several endogenous antioxidants (Demasi, A. P., Pcrcira, G. A., & Netto,

L. E., 2001). The role of lees surface in protecting wine from oxidation during sur lie aging should be then investigated on the entire cell (Martínez-Rodríguez, et al.,

2001).

However, the yeast cell wall contains more potentially antioxidant constituents

than just glucan (e.g. mannans and proteins). Therefore, the contribution of the

other cell wall components to the antioxidative activity was investigated too

(Jaehrig et al. 2008).

Cell wall degradation during autolysis is expected to affect the antioxidant capacity

of lees surface by improving the accessibility of the reactive groups and their

exposition to the medium (Gallardo-Chacón, J.J. et al., 2010).

However, the progressive loss of structural biomolecules (Pueyo et al. 2000) could

reduce the number of reactive groups. The balance between these phenomena

would determine the effective participation of lees surface in preventing wine

oxidation during sur lie aging.

Gallardo-Chacón J.J. et al., (2010) said that lees surfaces seem to progressively lose

their antioxidant activity the longer they are in contact with the wine. This leads to

the supposition that, subsequent to oxidation and the loss of structural

biomolecules, the effect of lower active group availability prevails over higher

accessibility due to weakening in the cell wall.

Inactive dry yeast (IDY) are commonly used in winemaking for several purposes.

They can be classified in four types, depending on the manufacturing process: 1

inactive yeasts (obtained by thermal inactivation of the yeasts and drying), yeast

Page | 20

autolysates (thermal inactivation followed by an incubation allowing enzymatic

activities and cell wall degradation), yeast hulls or walls (yeast walls without

cytoplasmic content), and yeast extracts (the soluble part of the autolysates, after

elimination of the cell walls) ( o o- ay n, et al. 2009, b).

The use of inactive dry yeast (IDY) delayed browning in wines, measured in terms

of the absorbance at 420 nm, the effect increase with the increase in yeast

concentration. The addition of IDY was also found to affect phenolic compounds,

particularly decreasing the concentrations of brown compounds (Lopez-Toledano,

A. et al., 2006).

Although the mechanism of interactions is unknown, some yeasts and yeast

derivatives have been found to retain anthocyanins in wine and to interact with

other polyphenols in model solutions (Lopez-Toledano A.,et al.,2006). In this

respect, some authors have proposed the use of yeast in wine fining treatments on

account of the selectivity of their cell walls to brown polymers (Bonilla, Mayen,

Merida, and Medina 2001);(Razmkhab et al., 2002). However, in addition to this

retention ability, yeasts in contact with model solutions of some flavans have been

found to delay browning, which have been ascribed to an inhibitory effect on the

formation of coloured compounds (Lopez- Toledano et al., 2002).

1.2.5. Combined effect of wine antioxidants

Sulphur dioxide in combination with ascorbic acid and glutathione The main preservative utilized in white wine to prevent oxidative spoilage is sulfur

dioxide (SO2). Often sulfur dioxide is used in combination with ascorbic acid,

because the latter can efficiently scavenge oxygen before reaction of oxygen with

phenolic compounds (Sonni,F. et al., 2011). If ascorbic acid is used without sulfur

dioxide, then the hydrogen peroxide and dehydroascorbic acid degradation

products can lead to the formation of spoilage pigments (yellow xanthylium

cations formed from the skin- and seed-derived flavonoid (+)-catechin) upon the

near depletion of ascorbic acid (Bradshaw, M. P. et al., 2003).

Another compound studied as complementary with SO2 is the tripeptide

Glutathione, γ-L-Glutamyl-L-cysteinylglycine(GSH). It has shown some potential to

assist sulfur dioxide in its role as an antioxidant. Certainly, glutathione is already

known to be capable of performing the main antioxidant reactions of sulfur

dioxide, although this has not always been in a wine-related matrix. Glutathione is

known to scavenge o-quinone compounds efficiently in wine/juice conditions. It

can react with hydrogen peroxide and undergo addition reactions with aldehyde

compounds, although these reactions have not been studied in wine-like

Page | 21

conditions. Some authors examined the oxidation of caffeic acid in the presence of

a variety of thiols, including glutathione, and showed a protective effect of the

thiols against both caffeic acid loss and oxidative color production (Cilliers, J. J. L.;

Singleton, V. L., 1990). However, this study was in alkaline aqueous solutions,

without added ethanol, metal ions, or (+)-catechin (Sonni F.et al., 2011).

Ascorbic acid with glutathione

Because ascorbic acid has a dual role in coloration reactions (protective vs.

enhancing), it had been examined the impact of glutathione on pigment

production in model wines both with and without ascorbic acid (Sonni F. et al.,

2011).

The ascorbic acid and glutathione in combination offers greater protection against

oxidative coloration when present together rather than in isolation. However, the

extent of protection was favored by the higher glutathione to ascorbic acid ratio

(Glutathione was utilized at concentrations 20-fold higher (860 mg/L) than the

normally found in wine to afford ascorbic acid (500 mg/L) to glutathione ratios of

1:1). Glutathione was able to delay ascorbic acid degradation and also inhibit the

formation of a known pigment precursor formed from an ascorbic acid

degradation product. Alternative caffeic acid degradation products were formed

from the glutathione/ascorbic acid combination that upon decay led to a diverse

range of polymeric pigments that were undefined.

It had been provided mechanistic insights into the role of glutathione, and the

glutathione/ ascorbic acid combination, during the storage of a model wine

system in oxidizing conditions. It was demonstrated the protective effect possible

with the antioxidants and their ability to change the range of pigments generated

along with the corresponding pigment precursors, (Sonni F. et al., 2011).

Page | 22

1.3. Aim of this work

On the basis of what was discussed, besides sulfur dioxide, different solutions

could be adopted to protect wines from oxidation; nevertheless, sulfites are still

considered not replaceable by any other additive. Moreover, the antioxidant

capacity of certain compounds has still to be well investigated: the activity of

glutathione, for example, has been well established on the musts, but in wine, very

few studies report useful results to confirm or not the possibility to use it to

partially replace sulfites.

For this reason, the aim of this work was to investigate the antioxidant behavior of

different additives and enological products in comparison with sulfur dioxide,

considering both their scavenging activity towards free radicals (DPPH) and their

ability to affect wine oxygen consumption rate and oxidizability, in order to

evaluate the possibility to use them in replacing or in reducing overall SO2 content

in wines. Trials have been performed on both model solution and wine.

Page | 23

2. Materials and Methods

2.1. Chemicals and oenological products

Potassium metabisulfite, sodium hydroxide, tartaric acid, 96 % v/v ethanol, 37 %

hydrochloric acid and sodium acetate were from Carlo Erba Reagents (Milan, Italy);

ascorbic acid, glutathione, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical and

HPLC grade methanol were from Sigma-Aldrich (St. Louis, MO, USA).

The inactive dry yeast was a thermal yeast autolysate, prepared as reported by

Comuzzo et al., (2012).

Yeast lees were obtained after alcoholic fermentation and racking of a white table

wine from Friuli Venezia Giulia region (Italy, harvest 2011).

2.2. Evaluation of the radical scavenging activity of different oenological

additives and products

To evaluate the radical scavenging activity of the previously mentioned antioxidant

products (yeast autolysate and lees) and compounds (sulfites, ascorbic acid and

glutathione) toward DPPH free radical, some trials have been performed both in

wine and in model solution.

2.2.1.Samples preparation

Model solution

For the trials performed in model buffer, wine-like solution was a tartaric

hydroalcoholic buffer (0,03 M tartaric acid, buffered at pH 3,2 with 10 M sodium

hydroxide). As concerns the three pure antioxidant compounds (potassium

metabisulfite, ascorbic acid and GSH) a stock solution (500 mg/L) was prepared

fresh daily, and then used (100 µL) for DPPH trials, at the following concentrations:

50 mg/L (dilution 1:10) and 500 mg/L (no dilution; not performed for ascorbic

acid). Regarding inactive dry yeast, a 2,5 % w/v suspension was prepared in model

buffer, and 100 µL was subjected to DPPH assay, as reported below. Finally, yeast

lees was diluted in model buffer (2,5 mL/100 mL) and then used for DPPH analysis.

Wine

Trials on model buffer were repeated on a white Table wine from Friuli Venezia

Giulia (Italy), vintage 2010; in this case a control wine was also included in the

experimental project.

Page | 24

All the experiments were carried out in three repetitions for both wines and model

solutions.

2.2.2. DPPH assay

The use of the stable free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) to estimate

antioxidant activity was performed by a modification of the methods reported by

Brand-Williams et al. (1995) and Gallardo-Chacón et al. (2010). Trials were made in

model solution and in white wine.

Briefly, the DPPH was dissolved in a 60:40 mixture of methanol and 0,1 M acetate

buffer (sodium acetate, buffered at pH 4,5 with 6 M hydrochloric acid) until the

absorbance of the solution (515 nm) was 0,700; this stock solution was prepared

fresh daily; 3 mL of DPPH stock solution was introduced in a 1 cm path length

glass cuvette. Fresh prepared antioxidant solutions (100 µL) were added, and DPPH

discoloration was followed at 515 nm during 10 min. Results were expressed as the

percent diminution of the original absorbance in 10 min [Abs 515 nm (%)].

For yeast lees and yeast extract, whose solubility is not complete, the reaction with

DPPH has been carried out in a test tube (3 mL of DPPH and 100 µL of yeast lees

or autolysate suspension, prepared as previously reported) for 10 min and then the

reaction mixture has been filtered on a 0,80 µm nylon membrane, before

spectrophotometric measurement (as suggested by Gallardo-Chacón et al., 2010).

2.3. Effect of different oenological additives and products on wine

oxygen consumption

2.3.1. Samples preparation

Two white wines were used respectively for two different set of trials: the first one

was a Friulano, D.O.C. Colli Orientali del Friuli (Italy), vintage 2007; the second was

a Chardonnay, D.O.C. Grave del Friuli (Italy), vintage 2011.

Each wine was used to fill 0,75 mL uncolored glass bottles, in which an Oxy2Dot®

sensor (OxySense Inc., Dallas, TX, USA) was previously introduced; samples were

vigorously mixed, until oxygen saturation was reached (temperature: 20 °C);

antioxidant compounds and products were then added and bottles were crown

capped and stored at 20-22 °C during all the time needed for the measurements

(two weeks).

Page | 25

All the trials were performed in three replicates; a control sample, without any

product addition was also prepared for each set of experiments. The amounts of

each antioxidant added for the two sets of trials are reported in

Table 1.

Table 1: Amounts of enological additives and products added in the two sets of

trials performed in section 2.3

Additive / product added

TRIAL 1

Control (no addition)

Sulfur dioxide 50 mg/L

Ascorbic acid 50 mg/L

Glutathione 50 mg/L

Yeast extract 500 mg/L

Yeast lees 2 % v/v

TRIAL 2

Control (no addition)

Sulfur dioxide 100 mg/L

Ascorbic acid (50 mg/L) + Sulfur dioxide

(50 mg/L)

Glutathione (50 mg/L) + Sulfur dioxide

(50 mg/L)

Ascorbic acid (50 mg/L) + Glutathione (50

mg/L)

Yeast Extract 1 g/L

Page | 26

2.3.2. Oxygen consumption measures

Oxygen levels (in mg/L) were measured inside the bottles filled with wines treated

with the different antioxidant agents, to establish how the products themselves

could affect wine oxygen consumption after bottling. The measures began

immediately after antioxidant addition and crown capping and then continued at

regular intervals (each hour during the first half a day and each day during two

weeks), as soon as the oxygen was almost totally consumed in one of the

treatments.

Dissolved oxygen was measured by means of an OxySense® fluorimeter

(OxySense Inc., Dallas, TX, USA –Figure 9); the method was based on the ability of

oxygen to quench the fluorescence of certain ruthenium (II) complexes.

Before filling the bottles with wine, an Oxy2Dot® sensor (OxySense Inc., Dallas, TX,

USA), on which surface a thin layer of one of such ruthenium complexes was

immobilized, was glued to the inner surface of the glass with a specific silicon

based oxygen permeable adhesive.

When this sensor is illuminated by a pulsed blue light ( 400-500 nm) emitted by a

led of the OxySense® equipment, ruthenium complex is excited, so that it emits a

red light by fluorescence ( 600 nm) when it returns to its ground state.

The oxygen measurement is based upon the quenching of such fluorescence by

oxygen. The presence of O2 quenches the fluorescent light from the complex as

well as its lifetime. The quenching process is a purely collisional dynamic where the

energy from the excited fluorescent dye is transferred to the oxygen molecule

during a collision, hence, reducing the emission intensity as well as the fluorescent

lifetime of the complex. The quenching is proportional to oxygen concentration

and the oxygen content is not changed by the measurement process.

Page | 27

Figure 9: OxySense® equipment to measure the oxygen consumption.

2.3.3. Spectrophotometric measurements

To evaluate eventual short-time effects of the tested products and additives on

wine color and oxidizability, some spectrophotometric measurements have been

performed on the wines after oxygen measurements (15 days after bottling).

Wine Color

The color of the wine was directly measured at 420 nm (1 cm path length glass

cuvettes), reading the absorbance against distilled water.

POM Test

The POM-test method was chosen for evaluating the oxidability of polyphenols

(Müller-Späth H., 1992). 5 mL of each sample were treated with 25 µL of a 3 %

hydrogen peroxide solution and immediately heated for 1 hour at 60°C. POM test

was expressed as the percent increase of color after peroxide addition, (Figure 10).

Figure 10: POM -test calculation

Page | 28

2.4. Statistical analysis

The results of the experiments were analyzed statistically using the software

Statistica for Windows, version 8.0 (StatSoft, Inc., Tulsa, OK, USA).

As concerns DPPH trials, One Way ANOVA was carried out on the Abs 515 nm

(%); means and standard deviations were calculated, and significant differences

were evaluated by Tukey HSD test, at p < 0,001.

To assess the differences in oxygen consumption rates, the curves obtained by

monitoring oxygen concentration were transformed as follows: at first, all data

were expressed in percentage respect to the initial O2 concentration; then,

logarithm (log10) was used to linearize the curves. Slopes and R2 have been

calculated and slopes were subjected to One Way ANOVA and Tukey HSD test, to

assess significant differences between oxygen consumption curves (p < 0,05).

Regarding spectrophotometric measures (Abs 420 nm and POM test values) and

final oxygen concentration in wines, One Way ANOVA and Tukey HSD test were

carried out as reported for DPPH assay; resulst were significant at p < 0,05.

Page | 29

3. Results and Discussion

3.1. Study of the behavior of some antioxidant agents in wine-like

buffer and in white wine using DPPH stable free radical.

3.1.1. Results of DPPH trials in wine-like buffer

In the first graph (Figure 11) we can observe the results of the measurements in

wine-like buffer. In this case, the Lees (2, 5%) showed the highest radical

scavenging activity in comparison to all the other treatments. Then, the Yeast

Extract (2, 5%) and SO2 (500mg/L) behaved similarly among them, having a

better antioxidant activity than the GSH (500mg/L) treatment. The latter behaved

similar to the Ascorbic acid (50mg/L) which behaved statistically similar than the

SO2 (50mg/L), both with the same concentration. Finally the GSH with the lesser

concentration (50mg/L) had the lesser antioxidant activity of all the treatments.

Figure 11: DPPH-test. Radical scavenging activity of different enological additives

and products, in wine-like solution: results of ANOVA analysis and Tukey HSD test;

different letters mark significant differences at p < 0,001

Mean ±SD Min-Max

b

d

a

b

c

e

cd

SO

2 5

00

mg

/L

SO

2 5

0 m

g/L

Le

es (

2,5

%)

Ye

ast E

xtr

act (2

,5 %

)

GH

S 5

00

mg

/L

GH

S 5

0 m

g/L

Asco

rbic

Acid

5

0 m

g/L

-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

a

bs

515

nm

(%)

Page | 30

As a summary of Figure 11 we can say that SO2 and Glutathione at the normal

levels found in wine ( 50mg/L), have the lowest radical scavenging activity, what is

more the Glutathione showed the lesser activity of all the treatments. Observing

these behaviour in comparision with yeast lees and yeast extract treatments; we

can see in the graph that we need ten times higher levels of sulfites (500 mg/L),

than the normal amounts found in wine, to simulate the antioxidant activity of

yeast extract. Anyway, the amount of extract used is higher than those normally

used in winemaking (lower than 1 g/L); the interesting thing is that lees antioxidant

capacity in model buffer, remains higher than the huge amounts tried of SO2 and

GSH (500 mg/L), this antioxidant capacity is due to lees polyphenols but also to

other molecules derived from yeasts. From this results we can say that probably

glutathione is not the only antioxidant compound present in the less to give them

this activity .

3.1.2. Results of DPPH trials in white wine

The same trials made in wine-like buffer were performed at this time on a white

table wine (from Friuli- Italy, vintage 2011). Even in this case, we can observe in

Figure 12, the Lees (2, 5%) treatment with the most radical scavenging activity

respect to the Control wine and the other treatments. Then, the Yeast Extract (2,

5%) together with the GSH (500mg/L) and Ascorbic acid (50mg/l) treatments

behaved statistically similar having more antioxidant activity than the other three

treatments: SO2 (500mg/L) , SO2 (50mg/L) and GSH (50mg/L) that behaved in

the same way, having the lesser activity as antioxidants in these trials. Naturally the

Control wine showed the lesser antioxidant activity respect to all the treated

samples with antioxidants.

Page | 31

Figure 12: DPPH- test. Radical scavenging activity of a table wine treated with

different enological additives and products: results of ANOVA analysis and Tukey

HSD test; different letters mark significant differences at p < 0,001

The behavior of some antioxidant agents in wine is different to what was observed

in wine-like buffer. In wine, the differences related to different amounts (50 or 500

mg/L) of SO2 and GSH found in wine-like solution seem less significant, probably,

wine components are also able to react with DPPH, leveling the effects of the

treatments. GSH and SO2 show the same effect (no significant difference),

contrarily to what was observed in wine-like buffer, where GSH was less effective in

discoloring DPPH (Figure 11).

Mean ±SD Min-Max

bb

a

ab ab

bab

c

SO

2 5

00

mg

/L

SO

2 5

0 m

g/L

Le

es (

2,5

%)

Ye

ast E

xtr

act (2

,5 %

)

GH

S 5

00

mg

/L

GH

S 5

0 m

g/L

Asco

rbic

Acid

5

0 m

g/L

Co

ntr

ol-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

a

bs

515

nm

(%)

Page | 32

3.1.3. Results of DPPH trials in white wine and the antioxidant

agents in pairs

These trials were performed on the same table white wine but sampled in a

different time and way. In this time we used some of the antioxidant agents in pair

and measurement at 515nm wavelengths were performed. We proved the pairs:

Ascorbic acid + Sulphur dioxide, Glutathione + Sulphur dioxide SO2 and

Glutathione +Ascorbic acid, all of them at a concentration of 50mg/L.

As we can see in the Figure 13, the wines treated with SO2 (50mg/L), Ascorbic

acid (50mg/L) and the couple SO2/Ascorbic acid (50mg/L) behaved statistically

similar to the Control showing the lesser antioxidant activity respecting to the

other treatments. Then, the wines treated with GSH (50mg/L) isolated and in pairs

(GSH/SO2 and GSH/Ascorbic acid), showed a better radical scavenging activity

respecting to the previous ones. Finally the treatments with Yeast lees (2, 5%) and

Yeast Extract (2, 5%) behaved as the best antioxidants among all the treatments.

Page | 33

Figure 13: DPPH test. Radical scavenging activity of different enological additives

and products isolated and in pairs in a table white wine: results of ANOVA analysis

and Tukey HSD test;different letters mark significant differences at p < 0,001

As summarize the result observed in Figure 13, we can say that the pairs did not

react as was expected and the yeast lees and yeast extract treatments behaved

very efficiently as radical scavengers. The Ascorbic acid, reacting alone or in pairs,

does not show significant differences respect to the Control and to the S02

(50mg/L), for that we can think that maybe the acid was already oxidized in the

stock solution. The GSH, alone and in pairs, behaved better than the Ascorbic

acid.

The pair GSH/Ascorbic acid had a higher radical scavenging activity than the acid

in combination with other antioxidants. In this case we can say that the GSH was

the compound that led the reaction, confirming that the Ascorbic acid had a lower

activity maybe due to a possible oxidation of the stock solution.

Also the GSH in this wine, in comparison with what occurred in the previous trial

(Figure 12), showed significative differences with the SO2 treatment, both at the

same concentration of 50 mg/L. This means that the wine influenced the activity of

the GSH. Depending of the wine composition, the activity of the GSH will be

different.

Mean ±SD Min-Max

c

aa

b

cc

b

c

b

SO

2 5

0 m

g/L

Le

es (

2,5

%)

Ye

ast E

xtr

act (2

,5 %

)

GH

S 5

00

mg

/L

GH

S 5

0 m

g/L

Asco

rbic

Acid

5

0 m

g/L

Co

ntr

ol

GH

S /S

O2

(5

0 m

g/L

)

AS

C/S

O2

(5

0m

g/L

)

GS

H/A

SC

(5

0m

g/L

)-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

0

A

bs 5

15

nm

(%

)

Page | 34

3.2. Effect of different oenological additives and products on wine

oxygen consumption capacity

FIRST SET OF TRIALS

In these first trials we used a white wine Friulano, D.O.C. Colli Orientali del Friuli

(Italy), vintage 2007. Figure 14 shows the oxygen concentration in milligrams per

litre (mg/L) versus the time (in hours). In this case, the fastest additives that

consumed almost all the oxygen were ascorbic acid (50mg/L) and yeast lees (2,

5%), they showed the highest oxygen consumption rate activity in comparison

with all the other treatments and the Control wine.

At the beginning yeast extract treatment was faster than the SO2, Control and

GSH ones but at the end of the measurements, the SO2 reached the lesser Oxygen

concentration. We can see that the Glutathione behaved very similar to the

Control wine, only having a little effect in oxygen consumption respect to it and

both having the lesser oxygen consumption in the time of measurements.

Page | 35

Figure 14: Oxygen consumption curves of a white wine (Friulano, D.O.C. Colli

Orientali del Friuli- Italy, vintage 2007) treated with different enological additives

and products. Means and standard deviations of three repetitions are reported.

The oxygen consumption was very fast in this trial. In futures proves it can be

useful to evaluate the content of some metals acting as catalysts, like copper and

iron, to understand better the trend of the curves. This general fast oxygen

consumption and the differences observed between the different antioxidants, in

comparison with the behavior of the antioxidant agents in the second set of trials

on young wine (see below in Figure 16) where we could not observed big

differences between the treatments, can be due to the state of the polyphenols in

the aged wine that maybe were already oxidized. So the performance of the curves

could be due only to the behavior of the antioxidant agent itself in the oxygen

consumption and, in a lesser extension, due to the polyphenols of this wine.

The graph in Figure 15 has been prepared as reported in Materials and Methods

(section 2.4. Statistical analysis). In the table 2 we have reported the equations of

the trend lines, their slopes and R2 values.

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

9,00

0 100 200 300 400

Ox

yg

en

co

nce

ntr

ati

on

(m

g/L

)

Time (hours)

Control

SO2 50 mg/L

Yeast extract 500 mg/L

Yeast lees 2 % v/v

Ascorbic acid 50 mg/L

Glutathione 50 mg/L

Page | 36

On the basis of these results we can confirm in Figure 15 what we observed in

Figure 14, having the yeast lees and ascorbic acid (distinct with “a”) as the best

antioxidants respect to the others which in this statistical analysis behaved in the

same way as a cluster (distinct with “b”).

Figure 15: One way ANOVA, carried out on the slopes of the lines obtained by

linearization (log 10) of the curves in figure 14. Means and standard deviations were

calculated and significant differences were assessed by Tukey HSD test at p < 0,05

Mean ±SD Min-Max

b b b

a

b

a

Contr

ol

SO

2 5

0 m

g/L

Glu

tath

ione 5

0 m

g/L

Yeast

lees 2

% v

/v

Yeast

extr

act

500 m

g/L

Ascorb

ic a

cid

50 m

g/L

-0,0140

-0,0120

-0,0100

-0,0080

-0,0060

-0,0040

-0,0020

0,0000

Slo

pe

(m

)

Page | 37

Table 2: Equations of the trend lines obtained by transforming (log 10) the curves

of oxygen consumption as detected for the different Friulano wine samples

analyzed in Figure 14 (average curves) and Figure 15 (ANOVA on slopes). Slopes

and R2 values are reported.

Sample Repetition log [O2 conc.] = -m (time) + q R2 Slope (m)

Control

1 y = -0,0043 x + 1,9689 0,9676 -0,0043

2 y = -0,0040 x + 2,0027 0,9632 -0,0040

3 y = -0,0040 x + 1,9713 0,9614 -0,0040

SO2 50

mg/L

1 y = -0,0040 x + 1,9989 0,9724 -0,0040

2 y = -0,0039 x + 1,9576 0,9787 -0,0039

3 y = -0,0045 x + 1,9685 0,9814 -0,0045

Glutathione

50 mg/L

1 y = -0,0047 x + 2,0148 0,9593 -0,0047

2 y = -0,0043 x + 1,9660 0,9557 -0,0043

3 y = -0,0035 x + 1,9622 0,9706 -0,0035

Yeast lees 2

% v/v

1 y = -0,0052 x + 2,0766 0,9214 -0,0052

2 y = -0,0079 x + 1,9130 0,9255 -0,0079

3 y = -0,0091 x + 1,9200 0,9657 -0,0091

Yeast

extract 500

mg/L

1 y = -0,0045 x + 1,9709 0,9613 -0,0045

2 y = -0,0039 x + 1,9780 0,9399 -0,0039

3 y = -0,0033 x + 1,8989 0,8675 -0,0033

Ascorbic

acid 50

mg/L

1 y = -0,0089 x + 1,9730 0,9943 -0,0089

2 y = -0,0095 x + 1,9727 0,9937 -0,0095

3 y = -0,0097 x + 1,9896 0,9974 -0,0097

Page | 38

Table 3: Oxygen concentration, DO 420nm and POM test detected for the Friulano

wine at the end of the measures (two weeks of bottle storage). Different letters

represent means which are significantly different (one way ANOVA and Tukey HSD

test) at p < 0,05

Sample

Oxygen concentration

(mg/L)

Mean + St. Dev.

Ascorbic acid 50 mg/L 0,13 + 0,01 a

Control 0,93 + 0,16 c

Glutathione 50 mg/L 0,81 + 0,19 c

SO2 50 mg/L 0,63 + 0,17 bc

Yeast extract 500 mg/L 0,89 + 0,32 c

Yeast lees 2 % v/v 0,14 + 0,02 ab

Sample DO 420 nm

Mean + St. Dev.

Ascorbic acid 50 mg/L 0,198 + 0,001 a

Control 0,204 + 0,002 a

Glutathione 50 mg/L 0,205 + 0,001 a

SO2 50 mg/L 0,182 + 0,002 a

Yeast extract 500 mg/L 0,206 + 0,024 a

Yeast lees 2 % v/v 0,273 + 0,010 b

Sample P.O.M. test

Mean + St. Dev.

Ascorbic acid 50 mg/L 42 + 1 a

Control 35 + 15 a

Glutathione 50 mg/L 42 + 5 a

SO2 50 mg/L 69 + 5 b

Yeast extract 500 mg/L 43 + 14 ab

Yeast lees 2 % v/v 30 + 9 a

The final effects of the treatments on wine composition have been evaluated after

a short-time storage period (two weeks). Observing the data, we can see that

oxygen concentration reflects what was already told in the previous pages.

Color measurements at 420 nm wavelength, demonstrated that only the treatment

with Lees behaved as significantly different to the others. The higher color

measured for the lees added wine, can be determined just by the color of the

Page | 39

added lees itself, but more probably just the higher oxygen consumption observed

for the lees treated samples could have determined this change in color.

The POM-test showed in this case that the different treatments are statistically

similar, only the SO2 treatment behaved as statistically different.

Talking about the mean values we can see that the Yeast lees treatment showed

the lowest value. Also this treatment obtained the lowest oxygen concentration at

the end of the 15 days of measurements (0,14mg /L) among all the treatments.

This low value means that the wine is the more stable in terms of polyphenols

oxidability, but looking at the color, we can also say that some oxidations could

have already occurred.

The wine treated with SO2 (50mg/L) showed the highest POM- test value; we

could say that the polyphenols in this case were well protected by the SO2 and

after the addition of the H2O2, a higher amount of polyphenols were available to

be oxidize and this increased the measured value. In effect, POM test is typically

higher in wines produced by hyper-reductive technologies.

Is very probable that these results are due to the age of the wine, which in this

case was an aged white wine as we already told. Regarding that the POM-test

must consider the type of wine, indeed it is very probable that a young wine would

be more oxidisable, obtaining higher POM-values, than an aged one; the

hydrogen peroxide would have an exclusively paling effect in the latter rather than

oxidant (E.Celotti et al.,2006). In effect, the SO2 treatment obtained the higher

POM-test value and the lesser Color value.

SECOND SET OF TRIALS

Figure 16 shows the Oxygen concentration in milligrams per litre (mg/L) versus the

time (in hours). In this case we worked with a young white wine (Chardonnay,

D.O.C. Grave del Friuli – Italy, vintage 2011). We can observe that the different

treatment behaved very similarly among them in comparison with the previous

aged wine where we could observe differences among the different antioxidant

agents. Also the behavior was slower than in the aged wine.

We are in front of two very different wines in terms of vintage, variety and

winemaking process to make comparisons (Figure 14 and Figure 16). We can

observe these differences and we can make some hypothesis of the situation. We

were expecting a different behavior for the second set of trials using the young

Page | 40

wine. We expected a faster reaction and bigger differences among the treatment

but it did not happened. We can suppose that the aged wine had already the

polyphenols with a degree of oxidation so the behavior of the curves in figure 4

was due to the antioxidants themselves and in a lesser extension due to the

polyphenols for that we could observed this differences among the treatments. In

the second young wine, having more antioxidant support due to a supposed

bigger amount of polyphenols we could not see significantly differences in the

results.

It must be useful in the future to performed further analyses in the wines in terms

of metals (copper and iron) and polyphenols content to understand better the

results.

Figure 16: Oxygen consumption curves registered in a white wine ( Chardonnay,

D.O.C. Grave del Friuli- Italy, vintage 2011) treated with different enological

additives and products. Means and standard deviations of three repetitions are

reported for each point.

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

9,00

0 100 200 300 400

Ox

yg

en

co

nc

en

tra

tio

n (

mg

/L)

Time (hours)

Control

SO2 100 mg/L

Ascorbic acid (50 mg/L) + SO2 (50 mg/L)

Glutathione (50 mg/L) + SO2 (50 mg/L)

Ascorbic acid (50 mg/L) + Glutathione(50 mg/L)

Yeast Extract 1 g/L

Page | 41

The graph in Figure 17 has been prepared as reported in Materials and Methods

(section 2.4. Statistical analysis). In the table 4 we have reported the equations of

the trend lines, their Slopes and R2 values.

In the Figure 17 we can confirm what was shown in Figure 16, where we could not

practically see differences among the treatments.

Figure 17: One way ANOVA, carried out on the slopes of the lines obtained by

transformation (log 10) of the curves in Figure 16. Means and standard deviations

were calculated and significantly differences were assessed by Tukey HSD test ( p <

0,05)

Mean ±SD Min-Max

b ab ab aab ab

Contr

ol

SO

2 1

00 m

g/L

Ascorb

ic a

cid

+ S

O2

Glu

tath

ione +

SO

2

Glu

tath

ione +

Ascorb

ic a

cid

Yeast

Extr

act

1 g

/L

-0,0140

-0,0120

-0,0100

-0,0080

-0,0060

-0,0040

-0,0020

0,0000

Slo

pe

(m

)

Page | 42

Table 4: Equations of the trend lines obtained by transforming (log 10) the curves

in Figure 16 (average curves) and figure 17 (ANOVA on slopes). Slopes and R2

values are reported

Sample Repetition log [O2 conc.] = -m (time) + q R2 Slope (m)

Control

1 y = -0,0012 x + 2,0121 0,9764 -0,0012

2 y = -0,0015 x + 1,9920 0,9891 -0,0015

3 y = -0,0014 x + 1,9398 0,9085 -0,0014

SO2 100 mg/L

1 y = -0,0015 x + 1,9920 0,9925 -0,0015

2 y = -0,0019 x + 1,9745 0,9767 -0,0019

3 y = -0,0016 x + 1,9909 0,9774 -0,0016

Ascorbic acid (50

mg/L) + SO2 (50

mg/L)

1 y = -0,0019 x + 2,0346 0,9618 -0,0019

2 y = -0,0017 x + 2,0217 0,9797 -0,0017

3 y = -0,0021 x + 2,0192 0,9886 -0,0021

Glutathione (50

mg/L) + SO2 (50

mg/L)

1 y = -0,0024 x + 2,0198 0,9215 -0,0024

2 y = -0,0019 x + 2,0524 0,9470 -0,0019

3 y = -0,0020 x + 2,0233 0,9807 -0,0020

Ascorbic acid (50

mg/L) + Glutathione

(50 mg/L)

1 y = -0,0017 x + 2,0242 0,9773 -0,0017

2 y = -0,0017 x + 1,9957 0,9904 -0,0017

3 y = -0,0013 x + 1,9786 0,9707 -0,0013

Yeast Extract 1 g/L

1 y = -0,0019 x + 2,0100 0,9210 -0,0019

2 y = -0,0015 x + 2,0152 0,8819 -0,0015

3 y = -0,0014 x + 1,9934 0,9809 -0,0014

Page | 43

Table 5: Oxygen concentration, DO 420nm and POM test detected for the wines at

the end of the measures (two weeks). Different letters represents means which are

significantly different (one way ANOVA and Tukey HSD test) at p < 0, 05

Sample

Oxygen concentration

(mg/L)

Mean + St. Dev.

Control 0,92 + 0,37 a

SO2 100 mg/L 0,79 + 0,05 a

Ascorbic acid (50 mg/L) + SO2 (50 mg/L) 0,64 + 0,07 a

Glutathione (50 mg/L) + SO2 (50 mg/L) 0,61 + 0,22 a

Ascorbic acid (50 mg/L) + Glutathione (50

mg/L) 0,77

+ 0,19 a

Yeast Extract 1 g/L 1,54 + 0,22 b

Sample DO 420 nm

Mean + St. Dev.

Control 0,080 + 0,001 b

SO2 100 mg/L 0,079 + 0,000 b

Ascorbic acid (50 mg/L) + SO2 (50 mg/L) 0,078 + 0,001 b

Glutathione (50 mg/L) + SO2 (50 mg/L) 0,079 + 0,001 b

Ascorbic acid (50 mg/L) + Glutathione (50

mg/L) 0,078

+ 0,001 b

Yeast Extract 1 g/L 0,069 + 0,001 a

Sample P.O.M. test

Mean + St. Dev.

Control 52 + 2 a

SO2 100 mg/L 53 + 1 a

Ascorbic acid (50 mg/L) + SO2 (50 mg/L) 47 + 16 a

Glutathione (50 mg/L) + SO2 (50 mg/L) 52 + 4 a

Ascorbic acid (50 mg/L) + Glutathione (50

mg/L) 46

+ 12 a

Yeast Extract 1 g/L 57 + 1 a

We can see first, in table 5, the results of Oxygen concentration reached after 15

days of measurement with the OxySense® equipment. In terms of statistical

differences we can only see the yeast extract treatment as significantly different of

all the other treatments and the Control wine.

Page | 44

If we comment the results in terms of mean values, the Yeast extract treatment

showed the lesser antioxidant activity, slower than the Control wine, both showing

the lesser antioxidant activity. Then, we can observe the SO2 (100mg/L) and the

GSH/Ascorbic acid (50mg/L) treatments, behaving similarly with the medium

antioxidant activity. Finally the Ascorbic acid/SO2 (50mg/L) and the GSH/SO2

(50mg/L) pairs reached the lowest oxygen concentration which means the highest

antioxidant activity.

Regarding the Color measurement with the spectrophotometer, as was already

mentioned in the Introduction of this work, the IDY delayed browning in the wines

measured in terms of the absorbance at 420 nm, the effect increase with increasing

in yeast concentration (Lopez-Toledano A. et al., 2006). In the second set of trials

was used the double IDY concentration and the measurement of the color showed

a slight difference in the color in the Yeast extract treatment respecting to the

others, we can see that this treatment with IDY resulted in a lower absorbance

value at 420 nm wavelength.

Regarding the POM-test technique, we could not observe big differences in the

results that behaved statistically similar. In terms of mean values, the

GSH/Ascorbic acid (50mg/L) and Ascorbic acid/SO2 treatments obtained the

lowest POM-value ( 46 and 47 respectively); this can means that these are the most

stable wines among the other treatments in terms of polyphenols oxidability. The

Yeast extract treatment behaved as the lesser stable wine having the higher POM-

value (57) this can means that this sample has already polyphenols to be oxidized.

Page | 45

4. CONCLUSIONS

Concerning the radical scavenging activity of the different oenological products

and additives tested, we have demonstrated that some of them showed a good

performance, compared with sulfur dioxide. Ascorbic acid, for example, was very

similar to sulfites, in all the tested conditions.

Yeast lees and yeast extract, in particular, had a good activity, even higher than

that detected for the average amounts of SO2 used in winemaking (50 mg/L);

higher sulfite concentrations (500 mg/L) are needed to have similar effects.

Concerning glutathione and its radical scavenging activity compared with SO2, it

did not have the same behavior in wine-like solution than in wine and also it

behaved differently in the different wines used. In wine-like solution it was less

active than SO2 (at the same concentration) in discoloring DPPH, while, in wine it

reacts in a similar way or even better. According to this observation, we can

conclude that the anti-radical action of both GSH and SO2 in wine, also depend on

wine characteristics and composition (level of quinones, acetaldehyde, etc.), so that

on the basis of these results, GSH could not be a potential replacement of sulfites

in all the situations. Obviously, we want to remark that, from this point of view, its

effects on wine composition have still to be evaluated.

The different behavior of the oenological additives and products was clearly

observable also concerning the oxygen consumption measurements. In the first set

of trials, the oxygen consumption was very fast. Yeast lees and ascorbic acid

significantly increased the oxygen consumption rate, respect to the other additives.

In the second set of trials, using a young wine, we had slower oxygen consumption

and less significant differences among the treatments; in these conditions, yeast

extract (IDY) gave the higher oxygen concentration at the end of the monitoring

period (two weeks). In both the performed experiments, IDY in particular, showed

faster oxygen consumption at the beginning of the monitoring time, becoming

slower later. It is difficult to find an explanation of this trend; what is clear is that

the ability of these additives to consume oxygen could be easily reduced with

contact time. In one case, IDY gave also positive effects on wine color protection.

Nevertheless, the storage time was probably too short to allow the evaluation of

significant effects of the different treatments on wine composition. Further trials

shall be performed on different wine types, with longer storage times, to have a

more complete set of results.

Page | 46

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ACKNOWLEDGMENTS

I have taken efforts in this project. However, it would not have been possible

without the kind support and help of many individuals and organizations.

First and foremost I am highly grateful to the EMaVE Consortium for gave me this

opportunity, since they selected me among many people to be part in the Vinifera

Euromaster programme.

I wish to express my gratitude to Prof. Enrico Peterlunguer and to my main

supervisor, Prof. Roberto Zironi, for welcoming me in the Food Science

Department of the University of Udine and giving me the opportunity to develop

this work. Special thanks for my co-supervisor, Dottore Piergiorgio Comuzzo, who

was abundantly helpful and offered me invaluable assistance, support and

guidance.

I would also like to thank Prof. Antonio Morata from the UPM (Universidad

Politécnica de Madrid) – España, for accepting to be part in the Commission of the

final examination of this Mater thesis.

Thank you very much to professors Emilio Celotti and Franco Batistuta for their

availability when it was required.

Finally I wish to express my infinite gratitude to my beloved family; for their

endless love and for encourage me every day through the duration of this

experience in Europe. Especially to my parents, who travelled more than 10.000 km

from Argentina to Udine, to be present in my Master thesis presentation.

My sincere thanks to all of them!

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