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Eur Food Res Technol (2008) 227:835–843
DOI 10.1007/s00217-007-0794-6
123
ORIGINAL PAPER
Antioxidant properties of traditional balsamic vinegar
and boiled must model systems
Davide Tagliazucchi · Elena Verzelloni · Angela Conte
Received: 11 July 2007 / Revised: 30 October 2007 / Accepted: 4 November 2007 / Published online: 21 November 2007
© Springer-Verlag 2007
Abstract Traditional balsamic vinegar (TBV) is a natu-
ral product prepared with cooked and concentrated locally
grown grape must. It has been demonstrated that TBV
contains phenols and shows antioxidant activity. In this
study we investigated the antioxidant properties of TBV
in relation to its content of phenolic compounds, poly-
meric tannins and Maillard reaction products (MRPs).
Results show that TBV has a high antioxidant activity
measured with both FRAP and ABTS assays, which is
higher or equal to those obtained in some red wines.
About 45% of the antioxidant activity of TBV is due to
the total polyphenolic fraction. Among polyphenols, tan-
nins contribute to about 50% of the antioxidant activity of
the total polyphenolic fraction. The residual antioxidant
activity of TBV is due to the melanoidins (about 45%)
synthesized during the boiling of the must and the ageing
of TBV and to other compounds such as low molecular
weight MRPs. When we investigated the eVect of heating
on the browning and on the formation of antioxidant
MRPs in must model systems, we observed a major for-
mation of antioxidant MRPs for the model system con-
taining both amino acids and sugars with respect to the
model system containing only sugars. We also tested the
eVect of some representative phenolic compounds present
in must. Only polyphenols were stable in the model
solution; however, at our experimental conditions they
did not inXuence the browning and the formation of
MRPs. Independent of their bioavailability, dietary anti-
oxidants play an important role in protecting the gastro-
intestinal tract from oxidative damage and also possibly
against a buildup of peroxides and their assimilitation in
the digestive tract.
Keywords Antioxidant activity · Maillard reaction ·
Melanoidins · Model solutions · Polyphenols · Tannins ·
Traditional balsamic vinegar
Introduction
Traditional balsamic vinegar (TBV) from Modena and
Reggio Emilia is a natural product with a large national
and international consumption, prepared from cooked
and concentrated locally grown grape must in wooden
barrels containing aged TBV, and with natural yeasts
providing alcoholic fermentation of sugars and acetobacters.
Trebbiano, or other white or red grapes grown in
Modena and Reggio Emilia provinces, supplies the grape
juice for the production of traditional balsamic vinegar.
This grape juice, also called must, is slowly concentrated
to at least three times the starting volume to produce the
cooked and concentrated must that represents the raw
starting material for making TBV [1]. The alcoholic fer-
mentation and acetic biossidation of cooked and concen-
trated must is performed in a set of barrels composed of
at least Wve wooden casks of diVerent volumes. Once per
year, the transfer of a volume fraction from the barrel
containing the younger vinegar to the older one is carried
out. Thus, from the barrel containing the vinegar in the
D. Tagliazucchi (&) · A. Conte
Department of Agricultural and Food Sciences,
University of Modena and Reggio Emilia,
Via Kennedy 17, 42100 Reggio Emilia, Italy
e-mail: davide.tagliazucchi@unimore.it
E. Verzelloni
Department of Human and Environmental Sciences,
University of Pisa, Via Roma 55, 56126 Pisa, Italy
836 Eur Food Res Technol (2008) 227:835–843
123
oldest ageing stage (barrel 1), a fraction is withdrawn and
bottled. Barrel 1 is Wlled up with the same volume of vin-
egar from the barrel at an earlier ageing stage (barrel 2).
This procedure is carried out until the barrel containing
the youngest vinegar (barrel 5) is reached. This barrel
will be integrated with the new cooked must [1]. The
Wnal result is a dark, thick and aromatic product with
high sugars and organic acids content [1, 2]. In addition,
TBV contains phenolic acids [3] and Xavonoids [4] and
shows a high antioxidant activity [4]. This antioxidant
activity is partially due to its phenolic content and to
other compounds present in TBV, such as compounds
synthesized during must cooking, as a result of caramel-
ization or of Maillard reaction.
The Maillard reaction occurring between an amino acid
or protein and a reducing sugar is a ubiquitous food reac-
tion that takes place during storage, cooking and heat pro-
cessing [5]. This reaction may produce a large number of
Maillard reaction products (MRPs) such as colorless aroma
compounds, ultraviolet absorbing intermediates and dark-
brown polymeric compounds called melanoidins [5].
The Maillard reaction plays an important role in the pro-
duction of some foods such as bakery products, coVee or
malt products. In these, the Maillard reaction is related to
the development of characteristic aroma, taste and color. In
addition, the Maillard reaction in amino acid–sugar model
systems [6–8] or protein–sugar model systems [9, 10] as
well as in food [11, 12] has been associated with the forma-
tion of compounds with strong antioxidant activity.
Several studies have investigated the formation of 5-
hydroxymethyl furfural (HMF) during the cooking of must
[13–16]. HMF can be formed by heat treatment of food
that is rich in sugars and is the main product of glucose
and fructose degradation in TBV. HMF is also a major
product of the Maillard reaction. However, as demon-
strated in our previous work [4], HMF does not show anti-
oxidant properties and, therefore, does not contribute to
the high antioxidant activity of TBV. It has recently been
demonstrated [15, 16] that the Maillard reaction can occur
during boiling and storage of grape juice and its model
system and results in the accumulation of HMF and brown
pigment formation.
Taking into account these observations, it is reasonable
to think that during the cooking of must, MRPs with antiox-
idant activity are synthesized and could be partially respon-
sible for the high antioxidant activity of TBV.
In this study we investigated the antioxidant properties
of TBV, in relation to its content in polyphenols, polymeric
tannins and MRPs, and the formation of antioxidant Mail-
lard reaction products during the heating of must model
systems with the aim of increasing the knowledge about the
formation of antioxidant compounds during the cooking
and concentration of grape must.
Materials and methods
Materials
L-arginine, L-glutamic acid, L-glutamine, L-proline, L-threo-
nine, catechin, gallic acid, bovine serum albumin (fraction
V), sodium dodecyl sulfate (SDS), triethanolamine, 4-
aminoantipyrine (4-AP), horseradish peroxidase (HRP)
type II and 2,4,6-tripyridyl-S-triazine (TPTZ) were sup-
plied from Sigma (Milan, Italy). 2,2⬘-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS) was supplied
from Calbiochem (La Jolla, CA, USA). All the other chem-
ical reagents were from Carlo Erba (Milan, Italy). Sepha-
dex C-18 columns (quantity of sorbent 500 mg, volume
above packing 6 mL, catalogue number 205350) were sup-
plied from Alltech (DeerWeld, IL, USA). Traditional balsa-
mic vinegar (TBV) samples were kindly supplied by the
“Consorzio fra produttori di Aceto Balsamico Tradizionale
di Reggio Emilia” (Reggio Emilia, Italy). The absorbance
was read using a Jasco V-550 UV/Vis spectrophotometer.
Extraction and determination of polyphenol
Polyphenolic compounds were extracted from three TBV
samples utilizing a previously developed protocol [4].
BrieXy, 2 mL of 10 times diluted vinegar (1 g of TBV
brought to 10 mL with distilled water) was passed through
a preconditionated Sephadex C-18 column. The columns
were washed thrice with 2 mL of water and the adsorbed
phenols were eluted thrice with 3 mL of methanol (HPLC
grade) on the basis of UV monitoring. At the end of the
separation, the methanolic fractions (M1, M2 and M3) con-
taining polyphenols were separately analyzed. These meth-
anolic fractions represent the total polyphenolic fraction of
TBV. The total phenolic content of the methanolic fractions
was determined using an enzymatic method [4, 17]. In a
3 mL spectrophotometric cell, 0.1 mL of appropriately
diluted methanolic fractions or catechin standard solutions
was added to 3 mL of 0.1 M potassium phosphate-buVered
solution, pH 8, containing 3 mM 4-AP, 2 mM H2O2, and
10 U of horseradish peroxidase (HRP). The absorbance
value was read at 500 nm at the endpoint of 15 min. Cate-
chin standard solutions were prepared by dissolving cate-
chin in water at a concentration ranging from 1 to 30 mg/L.
The total phenolic content was expressed in milligrams of
catechin equivalents per kilogram of TBV. The total pheno-
lic content was also determined with the Folin-Ciocalteu
reagent [18]. In a 1.5 mL Eppendorf tube, 790 L of dis-
tilled water, 10 L of appropriately diluted methanolic frac-
tions or catechin standard solutions and 50 L of Folin-
Ciocalteu reagent were added and mixed. A volume of
10 L of water or methanol was used as blank. After
exactly 1 min, 150 L of 20% aqueous sodium carbonate
Eur Food Res Technol (2008) 227:835–843 837
123
was added, and the mixture was mixed and left to stand at
room temperature in the dark for 120 min. Detection was
achieved at 760 nm. Catechin standard solutions were pre-
pared by dissolving catechin in water at a concentration
ranging from 50 to 500 mg/L. The total phenolic content
was expressed in milligrams of catechin equivalents per
kilogram of TBV.
Fractionation of phenolic compounds
Polyphenolic compounds were fractionated from three
TBV samples utilizing Sephadex C-18 columns [19].
BrieXy, 2 mL of 10 times diluted vinegar (1 g of TBV
brought to 10 mL with distilled water), adjusted to pH 7
with 5 N NaOH, was passed through a preconditionated
Sephadex C-18 column to adsorb neutral phenolic com-
pounds. Non-adsorbed phenolic acids were washed with
3 mL of water at pH 7. After acidiWcation of this eZuent to
pH 2 with HCl 0.1 N, phenolic acids were Wxed into a sec-
ond preconditioned acidic Sephadex C-18 column. The col-
umns were washed with 3 mL water at pH 2 to eliminate
interfering compounds and the adsorbed phenolic acids
(fraction I) were eluted with 2 mL of methanol (HPLC
grade). The fraction containing catechins and olygomeric
procyanidins (fraction II) was eluted with 2 mL of 16%
acetonitrile at pH 2 after acidic preconditioning of the Wrst
C-18 column. The Xavonols (fraction III) and polymeric
procyanidins (fraction IV) were eluted using 2 mL ethyl
acetate and 2 mL methanol (HPLC grade), respectively.
Polyphenols in all the fractions were determined with both
the enzymatic and Folin-Ciocalteu assays. Owing to the
diVerent responses of individual phenolic compounds in the
enzymatic method [20], we chose diVerent standards for
each fraction. Standards were: caVeic acid in methanol for
fraction I, catechin in 16% acetonitrile at pH 2 for fraction
II, quercetin in ethyl acetate for fraction III and catechin in
methanol for fraction IV. Each standard was tested at con-
centrations ranging from 50 to 500 mg/L. In Folin-Ciocal-
teu assay, catechin was used as standard compound.
Extraction and determination of tannin
Tannins were extracted from three TBV samples utilizing a
protein precipitation method [21]. BrieXy, 1 mL of 10 times
diluted TBV (1 g of TBV brought to 10 mL with distilled
water) was added to 2 mL of standard protein solution. The
solutions were mixed and allowed to stand at room temper-
ature for 15 min and were then centrifuged for 15 min at
5,000g. The standard protein solution consists of bovine
serum albumin dissolved at a concentration of 1 mg/mL in
0.2 M acetate buVer, pH 5, containing 0.17 M sodium chlo-
ride. After centrifugation, the supernatant was discarded
and the surface of the pellet was washed with acetate buVer
without disturbing the pellet. The precipitate was then dis-
solved in 4 mL of sodium dodecyl sulfate (SDS) triethanol-
amine solution that contained 1% SDS and 5% (v/v)
triethanolamine. This solution represents the tannic fraction
of TBV. The tannins were determined by mixing 2 mL of
tannic fraction with 0.5 mL of ferric chloride reagent
(0.01 M ferric chloride in 0.01 N HCl). The absorbance
value was read at 510 nm, approximately 15–30 min after
the addition of the ferric chloride reagent. Catechin stan-
dard solutions were prepared by dissolving catechin in
water at a concentration ranging from 5 to 100 mg/L. The
tannin content was expressed in milligrams of catechin
equivalents per kilogram of TBV.
Extraction of high molecular weight melanoidins
High molecular weight (>10 KDa) melanoidins were
extracted from three TBV samples as described by Morales
and Babbel [22]. DiVerent TBV samples of 1 g weight were
diluted to 10 mL with water and then Wltered (Whatman
Wlter papers 40, Whatman, UK). One aliquot (400 L) of
each Wltered sample was subjected to ultraWltration with
Microcon YM-10, regenerated cellulose 10 KDa (Milli-
pore, Italy) at 14,000g for 50 min at 4 °C. The retentate,
containing high molecular weight melanoidins and the
Wltrate containing low molecular weight compounds, such
as polyphenols and Maillard reaction products, were Wlled
up to 400 L with distilled water. The Wltrate was analyzed
for the total polyphenolic content with Folin-Ciocalteu
assay as described above.
Preparation of must model systems
Must model systems were prepared in accordance with
Bozkurt et al. [16]. To test the inXuence of sugars and
amino acids on the browning and on the production of anti-
oxidant compounds, two diVerent model systems were pre-
pared. Model system 1 was prepared to test the eVect of the
caramelization reaction and contained only sugars (98 g/L
fructose and 106 g/L glucose). Model system 2 contained
the same sugars as that of model system 1 and Wve amino
acids (1047 mg/L arginine, 449 mg/L proline, 210 mg/L
glutamine, 58 mg/L glutamic acid, 49 mg/L threonine). All
the model solutions were dissolved in water (50 mL) and
the pH was carefully controlled and adjusted to 3 with HCl.
The screw cap was kept loose to expose the must model
systems to air while heating in boiling water at 100 °C for
240 min, which is the time necessary to reduce the volume
to about 30% of the starting volume, simulating conditions
of must cooking. At diVerent times, 0.5 mL of the sample
was collected from must model systems and immediately
frozen until the analysis was performed. In addition, to test
the inXuence of the phenolic compounds on the browning
838 Eur Food Res Technol (2008) 227:835–843
123
and on the production of antioxidant compounds, two
diVerent model systems were prepared. Model system 3
was prepared to test the stability of the phenolic compounds
during heating to 100 °C. The phenolic compounds used
were gallic acid (as representative of phenolic acids) and
catechin (as representative of Xavonoids). We chose gallic
acid and catechin because they are easily separable and are
the most utilized polyphenol standards in literature. As
these belong to diVerent phenolic groups, they may present
diVerent behaviors during must cooking and Maillard prod-
ucts formation. The concentrations of gallic acid and cate-
chin were 17 and 35 mg/L, respectively, in accordance with
the value of total phenolic acids (hydroxybenzoic and cin-
namic acids) and total Xavonoids (procyanidins and Xavo-
nols) determined in white grape (Thompson seedless) must
by Spanos and Wrolstad [23]. Model system 4 contained
the same sugars and the same amino acids as that of model
system 2 and the same phenolic compounds of model sys-
tem 3.
Measurements of browning
The browning index of the three TBV samples, total poly-
phenolic fractions and high molecular weight melanoidins
extracted from TBV samples was determinate by measur-
ing the color as absorbance at 420 nm in a 1 cm glass
cuvette. The samples were appropriately diluted in water to
give absorbance values <1.
Measurements of antioxidant activity
Antioxidant activity of the three TBV samples, total poly-
phenolic fractions and high molecular weight melanoidins
extracted from TBV samples were measured both as reduc-
ing ability and radical scavenging activity. For the determi-
nation of the reducing ability of samples, a protocol based
on the ferric reducing/antioxidant power (FRAP) assay was
utilized [24]. BrieXy, 0.1 mL of diluted sample was added
to 3 mL of FRAP reagent that was freshly prepared by mix-
ing 300 mM acetate buVer, pH 3.6, 10 mM TPTZ in 40 mM
HCl, and 20 mM FeCl3 at a ratio of 10:1:1. After exactly
6 min, the absorbance was read at 593 nm. For the determi-
nation of the ABTS radical scavenging activity of samples,
a protocol based on the ABTS assay was utilized [25].
ABTS was dissolved in distilled water to 14 mM concen-
tration. ABTS radical cation (ABTS·+) was produced by
reacting ABTS stock solution at the ratio of 1:1 with
4.9 mM potassium persulphate and leaving the mixture to
stand in the dark at room temperature for 12–16 h before
use. The resulting blue-green ABTS radical solution was
diluted in ethanol to an absorbance of 0.700 §0.050 at
734 nm. A volume of 40 L of diluted sample was added to
1960 L of the resulting blue-green ABTS·+. The mixture,
protected from the light, was incubated in the Jasco V-550
spectrophotometer at 37 °C for 10 min; the decrease in
absorbance at 734 nm was measured at the endpoint of
10 min.
Vitamin C standard curves that correlate the concentra-
tion of vitamin C (ranging from 1 to 150 mg/L) and the
amount of absorbance reduction (ABTS scavenging assay)
or absorbance increase (FRAP assay), caused by vitamin C,
were obtained. The results were calculated as milligrams of
vitamin C per kilogram of TBV or milligrams of vitamin C
per liter of must model solutions.
Statistical analysis
All data are presented as mean §SD for at least Wve repli-
cations for each prepared sample. The statistical analysis
and regression analyses were performed using GraphPad
Instat (GraphPad Software, San Diego, CA, USA). DiVer-
ences of P< 0.05 were considered signiWcant. Correlation
between the antioxidant capacity and the browning index
was established using regression analysis at a 99% signiW-
cance level.
Results and discussion
All three samples of TBV show a high antioxidant activity
measured with both FRAP and ABTS assays (Table 1) that
is higher than or equal to that obtained in red wine [4, 26].
Table 1 Antioxidant activity, total polyphenolic content, tannins content and browning index of three diVerent samples of traditional balsamic
vinegar
Samples Antioxidant activity
FRAP (mg vitamin
C/Kg TBV)
ABTS (mg vitamin
C/Kg TBV)
Total polyphenols
(mg catechin/Kg TBV)
Tannins
(mg catechin/Kg TBV)
Browning index
(OD 420 nm)
TBV1 2,618.5 §115.8 4,236.4 §203.4 1,586.6 §41.8 250.2 §19.7 52.4 §3.4
TBV2 2,882.8 §132.6 6,915.2 §195.8 1,613.9 §34.1 328.0 §21.9 71.8 §1.0
TBV3 3,349.1 §150.0 6,630.2 §620.1 1,521.5 §88.6 343.3 §13.0 111.0 §3.9
Average 2,950.1 §369.9 5,927.3 §1471.3 1,574.0 §47.5 307.2 §49.9 78.4 §29.9
Eur Food Res Technol (2008) 227:835–843 839
123
The values obtained with the ABTS assay were higher
than those obtained with FRAP assay in each sample ana-
lyzed. The diVerence in the antioxidant activity obtained
with FRAP and ABTS assays could be due to the diVerent
reaction mechanism involved. FRAP assay detects com-
pounds that act only by the single electron transfer (SET)
mechanism, while ABTS assay detects compounds that act
either by direct reduction via the electron transfers or by
radical quenching via the hydrogen atom transfer (HAT)
mechanism [27, 28]. It is known that polyphenols present in
fruits, vegetables and related products such as red wine, tea
and vinegar are potent antioxidants and are responsible for
the main part of the antioxidant activity of these products.
As reported in Table 1, TBV also contains polyphenolic
compounds and polymeric tannins that contribute signiW-
cantly to its high antioxidant activity. The concentration of
unfractionated polyphenols of TBV, determined with per-
oxidase assay in the methanolic fractions after Sephadex
passage, was 1574.0 §47.5 mg catechin/kg TBV. Cate-
chin, which we have chosen as a reference compound, as
well as quercetin, apigenin and m-hydroxybenzoic acid, has
a middle molar absorbance () value with the peroxidase
method. Moreover, catechin is widely used in the Folin-
Ciocalteau assay. For these reasons, we have expressed the
values of the unfractionated phenols as catechin equivalent,
both for peroxidase and Folin-Ciocalteau methods. In the
literature [20], the of a number of compounds determined
by the peroxidase method is reported and it is easy to
express concentration with other standards. When polyphe-
nols were determined with the Folin-Ciocalteu assay, the
concentration was 2,205.4 §11.1 mg catechin/kg TBV,
higher than that determined with peroxidase assay. TBV
methanolic fractions may contain compounds that interfere
with Folin-Ciocalteu assay, mainly melanoidins that have
been shown to react in a concentration-dependent manner
with the Folin-Ciocalteu reagent [4]. For this reason, we
have eliminated from TBV the high molecular weight mel-
anoidins before C18 passage. When melanoidins were
eliminated, the total polyphenol concentration obtained
with Folin-Ciocalteu method in the methanolic fractions
were 1,882.2 §53.8 mg catechin/kg TBV closer to that
obtained with enzymatic assay.
When polyphenols are fractionated into phenolic acids,
Xavanols, Xavonols and polymers, the phenolic concentra-
tion was determined in each fraction with enzymatic assay
using speciWc standards. The phenolics most represented
were phenolic acids (37.8 §1.7% of the total polyphenols),
followed by catechins (36.0 §1.8%), polymeric procyani-
dins (18.8 §1.3%) and Xavonols (7.4 §1.5%). The total
phenolic content obtained from the sum of the concentra-
tions of each phenolic fraction was 1,398.3 §33.9 mg/kg
of TBV, which is similar to the value previously obtained
in the methanolic fractions. When the phenolic concentra-
tion was determined in each fraction with Folin-Ciocalteu
assay, the sum of the concentrations was 2,125.4 §
29.5 mg catechin/kg TBV, which is similar to the value
determined with Folin-Ciocalteu assay in unfractionated
poyphenols containing melanoidins. The value of tannins
obtained with the peroxidase assay after fractionation,
diVers slightly from the value obtained with the protein pre-
cipitation method (262.68 §18.2 mg catechin/kg TBV and
307.2 §49.9 mg catechin/kg TBV, respectively). This
diVerence could be probably due to an overestimation of
the concentration of tannins in the protein precipitation
method owing to the non-speciWc binding of catechin to the
protein–tannin complex [21], causing catechin precipita-
tion. On the other hand, when polyphenols are fractionated
[19], olygomeric procyanidins are recovered in fraction II
probably causing underestimation of the concentration of
tannins in fraction IV.
Phenolic compounds are already present in the must.
Antonelli et al. [13] found that uncooked must contains
about 260 mg/kg of polyphenols. Taking into account that
during the cooking the must is concentrated about 3.3
times, the concentration of polyphenols in concentrated and
cooked must should be 858 mg/kg. During the ageing, TBV
is concentrated about 1.8 times [2]; therefore, the amount of
polyphenols in aged TBV should be 1,544 mg/kg. This
value is close to the data obtained in our study, suggesting
that the increase in the concentration of polyphenols from
the uncooked must to the aged vinegar could only be due to
the concentration process. However, it is not possible to
exclude that other processes such as extraction from wood
or precipitation of the largest tannins could be important in
determining the concentration of polyphenols in aged TBV.
The cooking of must and the successive ageing of the
vinegar favor the Maillard reaction and the formation of
antioxidant MRPs. Absorbance at 420 nm represents the
browning index and is related to the brown pigment forma-
tion due to the Maillard reaction [11]. As can be seen in
Table 1, all three samples of TBV show a high browning
index that can suggest the presence of brown melanoidins.
However, some polyphenols such as Xavonols and Xava-
nol olygomers and polymers can themselves contribute to
the absorbance at 420 nm of TBV. In particular, the total
polyphenolic fraction of TBV accounts for the
42.9 §11.1% of the total absorbance at 420 nm (the aver-
age value obtained from Table 1 was deWned as 100% of
browning index). The residual value of absorbance at
420 nm is related to the presence of brown melanoidins.
As reported by Antonelli et al. [13], the browning index of
cooked must is about 3, while the browning index of aged
TBV observed in our samples is 78.4 (Table 1). This incre-
ment in the browning index is not explicable with the con-
centration of the product and indicates that the Maillard
reaction continues during the ageing of TBV, being
840 Eur Food Res Technol (2008) 227:835–843
123
favored by the reduction in water content [29] of the vine-
gar during the ageing.
To verify the eVect of the incidence of phenolic com-
pounds and tannins on the overall antioxidant activity of
TBV, we tested the total polyphenolic fraction and tannic
fraction extracted from the TBV samples for the antioxi-
dant activity assay. Phenolic compounds contribute to
47.4 §8.3 and 43.9 §6.2% of the overall antioxidant
activity of TBV measured by FRAP and ABTS assays,
respectively (Table 2).
Among polyphenols, tannins contribute to 45.3 §3.8
and 57.5 §2.7% of the antioxidant activity of the total
polyphenolic fraction measured by FRAP and ABTS
assays, respectively. The residual antioxidant activity of
TBV could be due to other compounds present such as
Maillard reaction products. To verify the eVect of the inci-
dence of melanoidins on the overall antioxidant activity of
TBV, we tested the high molecular weight fraction corre-
sponding to melanoidins extracted from the TBV samples
for the antioxidant activity assays. Melanoidins contribute
to 43.6 §2.3 and 46.4 §1.1% of the overall antioxidant
activity of TBV measured by FRAP and ABTS assays,
respectively (Table 2). The residual antioxidant activity
(about 10%) is due to other compounds such as low molec-
ular weight MRPs.
Numerous studies have shown the formation of MRPs
with antioxidant activity in model systems constituting a
sugar and an amino acid [6–8]. Generally, these studies
have been done without pH control [30–32] or at neutral [6]
or basic [7, 8] pH value. The pH has a great inXuence on the
kinetics of Maillard reaction; in fact, it is favored by high
pH values [33, 34]. At the same time, high pH values favor
the formation of melanoidins with high antioxidant activity
[11, 35]. Since pH has a great inXuence on the Maillard
reaction products with antioxidant activity [11], it is neces-
sary to match the pH value of the model systems to that of
the food being modeled.
The pH value of must is normally about 3; therefore, we
carried out the analysis to verify the formation of antioxi-
dant MRPs during the heating of must model systems with
a controlled pH value of 3. It has been demonstrated [13]
that during the cooking of must, the pH value is only
slightly aVected by the process with a recorded decrease of
0.3 pH units. Also in our model systems, the pH value
decreases by 0.3 §0.1 pH units. During heating, the vol-
ume of the must model solutions is progressively reduced
to about 80 (concentration ratio 1.25), 60 (concentration
ratio 1.64), 45 (concentration ratio 2.22) and 30% (concen-
tration ratio 3.33) of the starting volume after 60, 120, 180
and 240 min of heating, respectively.
Figure 1 shows the formation of brown pigment in must
model systems heated to 100 °C and pH 3.0, reporting the
uncorrected data for the concentration ratio and, therefore,
simulating the real conditions of cooking must.
As reported in Material and methods, model system 1
contained only sugars to verify the eVect of caramelization
on the total rate of browning, while model system 2 con-
tained both sugars and amino acids. The presence in the
reaction mixture of the amino acids caused an increase in
the browning of the model solution that is statistically
signiWcant (P< 0.05) with respect to the model system con-
taining only sugars. These data show that the non-
enzymatic browning during the heating of must model
system occurs through the formation of new compounds
synthesized either by caramelization or, in particular, by the
Maillard reaction and are in agreement with previously
reported data [16].
The models used in our study were selected according to
the major components of food responsible for the browning
reaction. However, the presence of minor components that
are not present in the model systems can inXuence the
browning reaction and presumably the formation of antiox-
idant compounds. For example, browning tends to be accel-
erated in the presence of metal ions [36] and inhibited by
salts [36, 37] and some organic acids [38]. All these
Table 2 Contribution of polyphenols and high molecular weight
(HMW) melanoidins on the total antioxidant activity of traditional
balsamic vinegar
aAverage value of three TBV samples as reported in Table 1
Samples FRAP (mg vitamin
C/Kg TBV)
ABTS (mg vitamin
C/Kg TBV)
TBV 2,950.1 §369.9a5,927.3 §1471.3a
Polyphenolic fraction 1,398.3 §244.9a2,602.1 §367.5a
HMW melanoidins 1,286.2 §67.9a2,750.3 §65.2a
Fig. 1 Brown pigment formation in must model systems at 100 °C
and pH 3.0; uncorrected data. Symbols used are: Wlled circle, model
system 1 (only sugars); Wlled triangle, model system 2 (sugars and
amino acids); Wlled square, model system 3 (only polyphenols); multi
symbol, model system 4 (sugars, amino acids and polyphenols)
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
Heating time (min)
0
OD 420 nm
50 100 150 200 250 300
Eur Food Res Technol (2008) 227:835–843 841
123
components are present in must and could have an inXu-
ence on the browning and on the formation of MRPs with
antioxidant activity.
In addition, the must contains a great quantity of pheno-
lic compounds that can be involved in the Maillard reaction
[11], for example interacting with HMF [39]. This interac-
tion has been demonstrated with catechin and results in the
formation of colored compounds [39]. However, the role of
phenolic compounds in the development of antioxidant
activity in heated sugar–amino acid systems has only
recently been considered [40, 41]. For example, it has been
demonstrated [41] that the incorporation of ferulic acid in
glucose–proline model system results in a signiWcant
increase in antioxidant activity as a consequence of the
reaction between ferulic acid and Maillard reaction prod-
ucts. Authors supposed that ferulic acid inhibits the free
radicals generated during the early stages of the Maillard
reaction, leaving MRPs with antioxidant activity to accu-
mulate [41]. To verify the possible role of phenolic com-
pounds present in the must on the browning reaction and on
the formation of MRPs with antioxidant activity, we carried
out experiments incorporating some polyphenols in the
must model systems. As shown in Fig. 1, no detectable
browning was observed after 240 min of heating to 100 °C
and pH 3.0 of model solution 3 containing only polyphe-
nols. The browning in model system 4 (containing amino
acids, sugars and polyphenols) was the same as that of
model system 2 (containing amino acids and sugars), sug-
gesting that polyphenols, in our experimental conditions,
do not inXuence the browning rate of the model system.
The browning value determined in the model system 4
(3.88 §0.3) is higher than the browning value observed
during the heating of real samples of must (about 3) [13].
This observation could be explained by the fact that the
must contains not only sugars, amino acids and polyphe-
nols, but also all those substances reported above that can
inXuence the browning rate of a real must sample.
In order to verify if the increment in the brown pigment
were a consequence of the concentration process or of the
formation of new compounds, we reported the data cor-
rected for the concentration ratio (Fig. 2). As can be seen in
Fig. 2, the browning rate exhibits the same trend as
reported in Fig. 1 for all the model systems considered.
Figure 3 shows the formation of antioxidant compounds
assayed by ABTS assay in must model systems heated to
100 °C and pH 3.0, reporting the data uncorrected for the
concentration ratio, and, therefore, simulating the real con-
dition of cooking must.
As can be seen, the heating of each model system caused
an increment in the antioxidant activity. The increment in
the antioxidant activity is higher for model system 2 than
model system 1 indicating that the Maillard reaction is the
most important cause that determines the formation of new
antioxidants in must model systems. Polyphenols tested in
our study have no eVect on the formation of antioxidant
compounds and the increase in the antioxidant activity
observed for the model system 3 was only a consequence of
the concentration process. The same trend was obtained
when the antioxidant activity was tested with the FRAP
assay (data not shown).
Figure 4 shows the formation of antioxidant compounds
in must model systems heated to 100 °C, and of pH 3.0,
assayed by ABTS assay, reporting the data corrected for the
concentration ratio, in order to verify if the increment in the
Fig. 2 Brown pigment formation in must model systems at 100 °C
and pH 3.0. Data were corrected for the concentration ratio as reported
in the text. Symbols used are: Wlled circle, model system 1 (only sug-
ars); Wlled triangle, model system 2 (sugars and amino acids); Wlle
d
square, model system 3 (only polyphenols); multi symbol, model
system 4 (sugars, amino acids and polyphenols)
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
0
Heating time (min)
OD 420 nm
50 100 150 200 250 300
Fig. 3 Antioxidant activity determined with ABTS assay in must
model systems at 100 °C and pH 3.0; uncorrected data. Symbols used
are: white bar, model system 1 (only sugars); light gray bar, model
system 2 (sugars and amino acids); dark gray bar, model system 3
(only polyphenols); black bar, model system 4 (sugars, amino acids
and polyphenols)
0
100
200
300
400
500
600
700
0
Heating time (min)
mg/L Vitamin C
60 120 180 240
842 Eur Food Res Technol (2008) 227:835–843
123
antioxidant activity were a consequence of the concentra-
tion process or of the formation of new compounds.
Heating of model system 1 containing only sugars
caused an increase of antioxidant activity that is related to
the browning (r= 0.9440). The presence of the amino acids
in model system 2 increased the antioxidant activity with
respect to the model system 1 (P< 0.05). The increase in
the antioxidant activity of the model system 2 with the heat-
ing time is related to the browning (r= 0.9801) suggesting
that the brown pigments synthesized either by carameliza-
tion or, in particular, by the Maillard reaction are responsi-
ble for the increment in the antioxidant activity observed in
must model systems. When the antioxidant properties of
the model system 3 were investigated, no variations were
observed as the heating time increased, suggesting that
there was no formation of new antioxidant compounds dur-
ing the heating of this model system. In addition, the data
reported for the model system 3 show that only polyphenols
are stable in the model system. A comparison of the antiox-
idant activity of the model system 4 with the sum of the
antioxidant activity of model system 2 and model system 3
shows that the values of antioxidant activity are identical.
This fact suggests that catechin and gallic acid at the con-
centrations considered in our study and under our experi-
mental conditions do not inXuence the formation of MRPs
with antioxidant activity. These results cannot exclude that
at higher concentrations or at diVerent pH value, catechin
and gallic acid inXuence the browning reaction and the anti-
oxidant activity of Maillard reaction products. The same
trend was obtained when the antioxidant activity was been
tested with the FRAP assay (data not shown).
From our results, it is clear that during the boiling of
must model systems, MRPs with antioxidant activity can
form. The brown pigment formation can continue during
the ageing in the vinegar barrel and is favored by the dehy-
dration of the product, so that at the end of the ageing the
concentration of brown melanoidins formed during cooking
of the must increased, explaining the high antioxidant
activity of TBV. Our results show that TBV has a high anti-
oxidant activity due to polyphenols and tannins and MRPs.
Independent of their bioavailability, dietary antioxidants
play an important role in protecting the gastrointestinal
tract from oxidative damage and also possibly against a
buildup of peroxides and their assimilitation in the diges-
tive tract [42]. In this contest, TBV, which is rich in antiox-
idants, is a seasoning that can contribute, along with
vegetables and wine, to an increase in the total amount of
antioxidants ingested during a meal.
Acknowledgments The authors thank Professor Paolo Giudici and
“Consorzio fra produttori di Aceto Balsamico Tradizionale di Reggio
Emilia” (Reggio Emilia, Italy) for the supply of traditional balsamic
vinegar samples.
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