Journal of Food Composition and Analysis 94 (2020) 103620

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Journal of Food Composition and Analysis

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Original Research Article

Chemical Characterization Of Commercial Balsamic Vinegar Glaze

Lisa Lancellotti a, Alessandro Ulrici b, Simona Sighinolfi a, Andrea Marchetti a, *
a
Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
b
Department of Life Sciences, University of Modena e Reggio Emilia, Via Amendola, 2, 42122 Reggio Emilia, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The present study reports the chemical characterization of commercial balsamic vinegar glaze (BVG), a side
balsamic vinegar reduction product of the world-famous Aceto Balsamico di Modena (ABM). BVG, originally a homemade sweet and sour
balsamic vinegar glaze sauce, is now produced on an industrial scale and is gaining a growing success. The determination of total
phenolic acids
titratable acidity, dry matter, ash, metals, sugars, phenolic acids and furfurals is reported for BVG samples of
metal content
different batches and years of production. Principal component analysis (PCA) and ANOVA test were applied to
glucose
fructose evaluate the experimental data. The results showed that the chemical characteristics of the product are almost
furfurals unchanged in the investigated years and well reflect the characteristics of the ABM. Moreover, it was found that
potentially harmful species, namely furfural compounds and heavy metals, were at a lower concentration than
that of homologous products. In particular, the concentration of heavy metals, such as Cu, Zn and Pb, suggests
that a preventive demetallization of the BVG starting raw materials occurs.

1. INTRODUCTION objective information. In particular, the determination of different

chemical parameters plays a key role for two distinct targets: the product
Nowadays, consumers are increasingly demanding and selective, characterization and the validation of the production process.
especially in the food field. Among the several factors that influence This study is intended to be a first step in the analysis of the balsamic
customer behavior, the attributes of a food product are relevant ele­ vinegar glaze (BVG), also commercially known as balsamic vinegar
ments in the decision-making process. In addition to the organoleptic reduction, sauce or cream. BVG is a side product of one of the most
qualities, ingredients, health and safety features, a link with a specific famous Italian condiments that represent food excellence in the world:
territory contributes to adding value to the food product. the Balsamic Vinegar of Modena, Aceto Balsamico di Modena (ABM), a
In this last context, since 1992 the European Union has protected Protected Geographical Indication (PGI) awarded product (Regulation
high-quality agricultural products based on their geographical origin (EC) No 583, 2009).
(Regulation (EU) No 1151, 2012). This protection is recognized through BVG is a sweet-and-sour dressing and represents a traditional home-
the introduction of quality marks, namely the protected designation of cooked preparation obtained by boiling down, in a small pan, ABM with
origin, PDO, the protected geographical origin, PGI, and the traditional sugar or honey, flour and different flavors, such as garlic, truffle, basil,
specialties guarantee, TSG, aimed at avoiding fraud and damage to the raspberry, etc., depending on personal taste, until a glaze is obtained.
consumers and producers as well. The promotion of food with specific Basically, a balsamic vinegar reduction is a concentrated and thickened
characteristics, the improvement of the income of farmers with a version of balsamic vinegar and many people choose the BVG as an
better-quality vocation and the promotion of traditional processes alternative to the most expensive Aceto Balsamico Tradizionale di
through innovations are the foundations of the European approach to Modena (ABTM) (Cocchi et al., 2002) or in the absence of an aged ABM
increase health, safety and overall quality of food products. (Regulation (EC) No 583, 2009). The main drawback of the
Therefore, to be competitive, the food industry mainly has to focus home-cooked preparation is the formation of potentially harmful com­
its efforts on the reliability of quality and safety rather than on quantity pounds. In fact, when foods with high sugar content are subjected to
and price. Based on these considerations, the chemical characterization thermal treatment a series of chemical modifications occur, such as the
of products, following holistic approaches able to support the “from formation of furan compounds, principally furan and 5-hydroxymethyl
farm to fork/table” concept, constitutes an effective tool to provide furfural (HMF) (Kowalski et al., 2013). Since the toxic effects on

* Corresponding author at: Department of Chemical and Geological Sciences, Via Giuseppe Campi 103, 41125, Modena, Italy
E-mail address: andrea.marchetti@unimore.it (A. Marchetti).

https://doi.org/10.1016/j.jfca.2020.103620
Received 14 March 2020; Received in revised form 17 June 2020; Accepted 14 August 2020
Available online 21 August 2020
0889-1575/© 2020 Elsevier Inc. All rights reserved.
L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

human health of furan and its derivatives are still controversial, the 99%, diethyl ether, methanol (MeOH), acetonitrile (ACN), HPLC grade,
monitoring of these species is always recommended (Glatt, Sommer, and reagent grade 0.1 N sodium hydroxide standard solution were
2006). purchased from VWR International (Milan, Italy).
In the last decades, the ABM-based glaze has found great success and,
to meet consumer preferences and expectations and market re­ 2.2. Sample collection
quirements, companies in the food sector are asked to develop new
forms of convenient food. Currently, the food industry provides a wide Twenty-four bottles of marketable unflavored BVG, coming from
range of differently flavored balsamic creams, that taste homemade but different batches, representative of the annual production of the years
are ready-to-use and packed in an easy-to-squeeze bottle. The com­ 2015, 2016 and 2017, were obtained directly from a local producer.
mercial BVG represent an economic product that combines practicality Samples were properly stored in a cool and dry place, away from direct
and connection to the territory, being a PGI-based product. sunlight, according to producer indication reported on the bottle, to
The industrial preparation is obtained by mixing, with the aid of avoid any degradation. Each bottle of BVG was homogenized before
special gear pumps, concentrated grape must, ABM (at least 20%), taking a sub-sample to be analyzed.
modified cornstarch (about 3%) and flavors. The presence of modified
cornstarch allows the formation of a gel with high viscosity that gives 2.3. Analytical methodologies
the product its typical creamy consistency (Tharanathan, 2005). Due to
its composition, BVG has chemical characteristics that are halfway be­ 2.3.1. Sugars
tween those of ABM and grape musts. Concerning the physical proper­ The sugars, glucose and fructose, were determined by liquid chro­
ties, such as viscosity and density, these are mainly regulated by the matography technique (OIV, 2019). In particular, a HPLC apparatus,
quantity and type of the modified cornstarch and the concentrated must equipped with a Beckman 116 solvent pump, a Beckman 406
added to the other ingredients (Saha & Bhattacharya, 2010). analog-digital interface, a Rheodyne 7025 injector with a 20 μL fixed
The advantage of the industrial production process, with respect to volume loop, a Shodex Sugar SH1011 column (300 mm x 8.0 mm i.d.)
the homemade preparation, is that the formation of carbonized sugars is thermostated at 34.0 ± 0.1 ◦ C and a HP 1047 refractive index detector
avoided or at least minimized, thus also the bitter taste that is usually thermostated at 40.0 ± 0.1 ◦ C, was used. The separation was performed,
present in the home-cooked preparation is absent. Moreover, industrial in isocratic elution mode, at 1.25 mL/min with water as mobile phase.
production also guarantees a controlled acidity that derives from the Samples were diluted about 1000 times by weight with deionized water
ABM, which increases the BVG conservation and healthiness of the and filtered, through a 0.22 μm hydrophilic membrane, prior to the
product. injection. Glucose and fructose were identified at 5.9 and 6.4 min,
Contrary to the provisions of the ABM, whose production process is respectively. Quantification was carried out by means of an external
regulated by a specific production rule (Regulation (EC) No 583, 2009), calibration curve accomplished for each measurement session. A
that limits to a well-defined value some chemical/physical parameters randomly selected BVG sample was used as a control sample and
(e.g. the density, total acidity and minimum amount of reducing sugars), measured several times over the different measurement sessions. The
to date the production of the marketable BVG is not subjected to similar standard deviations calculated for glucose and fructose in this sample
rules. Furthermore, while ABM and ABTM have been extensively studied were then associated with all samples.
(Caligiani et al., 2007; Consonni et al., 2008; Lalou et al., 2015; Lem­
metti & Giudici, 2011), to date no studies have been reported in the 2.3.2. Furfurals
literature about the chemical analysis of BVG. For these reasons, this Furfurals were determined by HPLC equipped with a Gynkotek
study represents a first approach to the characterization of the market­ HP580Bio pump, a Rheodyne 7025 injector with a 20 μL fixed volume
able balsamic vinegar glaze, in terms of chemical composition and loop, a Biorad column (250 × 4.6 mm, 5 μm particle size) C18 reversed-
production monitoring. phase, thermostated at 25 ± 0.1 ◦ C, and a Gynkotek 340S UV-Vis diode-
Our attention was focused on the determination of the following array detector (scan range set at 190-600 nm). The separation was
analytical parameters: sugars, furfurals, phenolic acids, dry matter, ash, performed, in isocratic elution mode, at 0.9 mL/min using a mixture of
total titratable acidity and metals content, that typically represent the water-ACN (10:90), both containing 0.07% (v/v) phosphoric acid, as
main components of the raw materials used in the BVG formulation. In mobile phase. The number of compounds belonging to the furfural
particular, the aim of this first study on BVG chemical composition was family that are present in BVG is very large. Identification was carried
to evaluate the effect of the variability of the raw materials on the out for 5-hydroxymethylfurfural (5-HMF), furfural, furfuryl alcohol and
chemical composition of the final product. To better focus on this aspect, 2-furoic acid, while quantification was limited to 5-HMF and furfural,
we have limited our study to a single producer from the province of since the others were lower than the limit of quantification. In partic­
Modena (Italy), investigating the chemical composition of 24 market­ ular, furan derivatives detection was carried out at 285 nm (5-hydrox­
able BVG samples, coming from different production batches, produced ymethylfurfural, tR=6.9 min), 277 nm (furfural, tR=13.1 min), 252 nm
over three years. One-way ANOVA and Principal Component Analysis (2-furoic acid, tR=11.6 min) and 217 nm (furfuryl alcohol, tR=9.5 min).
(PCA) were used to estimate the variability of the samples, focusing both Samples were diluted about 500 times by weight with deionized water
on the single analytical parameters and on the chemical composition of and filtered, through a 0.22 μm hydrophilic membrane, before injection.
the samples as a whole. Quantification was carried out by means of an external calibration curve
accomplished for each measurement session. A randomly selected
2. MATERIALS AND METHODS sample was used as a control sample and measured several times over
the different measurement sessions. The standard deviations calculated
2.1. Chemicals for the species determined in this sample were then associated with all
samples.
High purity deionized water, type 1, was obtained from a Milli Q
Element system (Millipore, Bedford MD, USA). Physical and chemical 2.3.3. Phenolic acids
parameters for Type 1 water comply with ASTM and ISO 3696 grade Phenolic acids were determined by liquid chromatography with a
purity specification (ASTM D1193, 1999). HPLC equipped with a Gynkotek HP580Bio pump, a Rheodyne 7025
All standards were obtained from Sigma Aldrich (Milan, Italy), with injector with a 100 μL fixed volume loop, an Atlantis C18 column (250 ×
a purity greater than 99%. 4.6 mm, 5 μm particle size) and C18 guard column, thermostated at 25 ±
Phosphoric acid, trifluoracetic acid (TFA), with a purity greater than 0.1 ◦ C, and a Gynkotek 340S UV-vis diode-array detector (scan range set

2
L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

at 190-600 nm). The separation was performed with linear gradient 2.3.4. Dry matter and ash measurement
elution using a binary solvent system of 0.1% (v/v) TFA in water (A) and Aliquots of about 4 g of sample were weighed into quartz crucible
0.1% (v/v) TFA in ACN (B) and detection at 280 nm (Salvatore et al., and subjected to heat treatment for seven days in an ISCO oven (Milan,
2013). Instrumental separation conditions are reported, as supplemen­ Italy) set at 104 ± 2 ◦ C. Dried samples were then weighted to determine
tary material, in Table 1S. The chromatographic determination of dry matter content. Ash measurement was carried out by means of a
phenolic acids was carried out after analyte extraction by means of Nannetti Kiln HB muffle furnace (Faenza, Italy) set at 600 ± 25 ◦ C. The
solid-phase extraction (SPE). dried samples, coming from dry matter determination, were subjected to
Extraction of phenolic acids was carried out using Discovery DPA-6S high-temperature treatment for 24 h. All sample measurements were
cartridges (500 mg, polymeric reversed-phase, Supelco, Milan, Italy). repeated twice.
The cartridge was first activated with 4 mL of MeOH followed by 2 mL of
deionized water, then 1 mL of 10 times diluted sample was loaded. The 2.3.5. Total titratable acidity
cartridge was then washed with 2 mL of water and the unbounded eluted Total titratable acidity (TA) was determined by pH metric titration
fraction discarded. Elution of phenolic acids was achieved by washing with a Denver Instrument, 215 pH-meter equipped with a combined
the cartridge with 4 mL of MeOH followed by 6 mL of diethyl ether. Schott glass electrode (OIV, 2019). For TA determination, 1 g of sample
The phenolic acid fraction was then evaporated to dryness, under N2 was weighed and titrated with 0.1 N NaOH. The titration endpoint was
stream, and re-dissolved with 1 mL of water-MeOH (80:20). Samples mathematically determined by means of first and second derivative
were filtered through a 0.22 μm syringe filter prior to the HPLC techniques. All the samples were measured in triplicate.
injection.
Quantification of the phenolic species was carried out for the 2.3.6. Metals
following compounds: gallic acid (tR = 15.8 min), protocatechuic acid Metal content was determined by means of atomic absorption spec­
(tR = 20.3 min), caffeic acid (tR=27.0 min), syringic acid (tR = 27.2 troscopy (AAS) (OIV, 2019). In particular, Li, Na, K, Mg, Ca, Fe, Cu and
min), and p-coumaric acid (tR=31.9 min). Caffeic and syringic acids Zn, were determined by means of a FS220 flame atomic absorption
were quantified together as syringic acid equivalent, due to a partial spectrometer (FAAS), equipped with a SIPS10 sample introduction
overlap of the respective chromatographic signals. Samples were pump system, both supplied by Agilent (Milan, Italy). While, Pb and Mn,
analyzed in duplicate and quantified by means of an external calibration were determined by means of a Perkin Elmer SIMAA6000 graphite
curve accomplished for each measurement session. furnace spectrometer (GFAAS), equipped with a dedicated Zeeman
Fig. 1 shows an example of the HPLC separation carried out on a background correction system (Milan, Italy). FAAS and GFAAS instru­
mixture of standard phenolic compounds, plot A, and on a BVG sample, ment parameters are reported, as supplementary material, in Table 2S
plot B. and 3S, respectively. All the samples were measured twice.

2.3.7. Limits of detection and quantification

Both the detection limit (LOD) and quantification limit (LOQ) were
determined based on the method described by Miller & Miller (2010). In
particular, the LOD was determined as the analyte concentration giving
a signal equal to the blank signal, yB, plus three standard deviations of
the blank, SB; while LOQ was calculated using the formula: yB+10SB. In
Table 4S, reported as supplemental material, are reported LOD and LOQ
values in addition to the working calibration ranges.

2.4. Data analysis

One-way analysis of variance (ANOVA) and the Tukey multiple

comparison test were used to check for significant differences (P = 95%)
among the three years of production. Moreover, in order to obtain a
general overview of the collected data, by exploring the contribution of
all the considered parameters to the variability of the analyzed samples,
principal component analysis (PCA) was applied.
Since variables had different scales and units, PCA was calculated on
autoscaled variables. Autoscaling consists in transforming each variable
by subtracting its average value and then dividing it by its standard
deviation. This transformation allows the data to be translated at the
origin of the reference system since each variable will have an average
value equal to zero, and also makes the variability of each variable
equally important in the construction of the PCA model, since each
variable will have a standard deviation equal to one (Wise and Gal­
lagher, 1996). One-way ANOVA and Tukey multiple comparison test
were performed using the Statistics and Machine Learning Toolbox (ver.
11.2, The MathWorks Inc., Natick, MA, USA), while the PCA model was
calculated using the PLS Toolbox (ver. 8.6, Eigenvector Research Inc.,
Wenatchee, WA, USA); both the Toolboxes run in the Matlab environ­
ment (ver. 9.3, The MathWorks Inc., Natick, MA, USA).
Fig. 1. A, Chromatographic separation of a phenolic acid standard mixture. 1.
Gallic acid; 2. 5-HMF; 3. Protocatechuic acid; 4. (+)-Catechin; 5. Chlorogenic 3. Results and discussion
acid; 6. Caffeic acid; 7. Syringic acid; 8. Vanillin; 9. p-Coumaric acid; 10. Ferulic
acid; 11. Myricetin; 12. 4-Dimethylaminocinnamaldehyde. B, chromatographic All the experimental results for individual samples are reported, as
separation of a real BVG sample. supplementary material, from Tables 5S to 9S.

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L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

Table 1 reports the average values of the analytes measured in the concentration in BVG was relatively low in comparison with homolo­
balsamic vinegar glazes, for the three years of production, together with gous products, such as PGI ABM and PDO traditional balsamic vinegar of
the one-way ANOVA and the Tukey multiple comparison test results. Modena (ABTM), for which 5-HMF values often exceed 5000 mg/kg
(Manzini et al., 2011; Masino et al., 2008). In any case, the low acidity of
3.1. Sugars the product, about 2% w/w, helps to maintain the 5-HMF values as low
as possible. Even if no statistically significant differences were high­
Sugars, namely glucose and fructose, represent the main components lighted among the three years of production, a general decreasing trend
of the glaze. The presence of the two monosaccharides is due to both the of the average values was observed from 2015 to 2017 (Table 1). This
concentrated grape must, that typically contains about 700 g/kg of could be ascribed to the fact that the improvement of the technological
sugars, and the ABM, where reducing sugars concentration must not be processes for the production of musts: namely, the use of multiple effects
less than 110 g/L (Regulation (EC) No 583, 2009). evaporating towers operating at low temperature and high vacuum
The average concentration of glucose and fructose over the three processing conditions, was such as to reduce the formation of these
considered years was approximately 220 and 227 g/kg, respectively, species.
and their ratio (glu/fru) was approximately equal to 0.97.
Concerning the statistical evaluation of the data, one-way ANOVA 3.3. Phenolic acids
showed a statistically significant difference among the years for both
glucose and fructose. The Tukey multiple comparison tests highlighted a In addition to the well-recognized protective role of phenolic acids
decreasing trend with increasing years for both the sugars, with signif­ on human health (Kumari, 2012), the investigation of these species is
icant differences between 2015 and 2017 (Table 1). As for the samples also related to the sensory quality of food. In fact, these compounds play
from 2016, while glucose showed intermediate values with no statistical an important role in several sensory properties, such as color, flavor,
differences with respect to 2015 and 2017, the mean value of fructose astringency and hardness, of food products like wine and wine vinegar.
was comparable to 2015 and different from 2017. Considering that the Phenolic acids composition is affected by grape quality and oeno­
sugar variation involves both glucose and fructose, a plausible expla­ logical practices (Cerezo et al., 2008, 2010; Van Leeuw et al., 2014). The
nation for this evidence might be a variation of the starting raw material presence of phenolic acids in wine and vinegar is mainly due to their
used to produce grape must and ABM. natural content in grapes, as secondary metabolites of plants, and con­
The glucose/fructose ratio did not show statistically significant tent is increased if the grape-derived products are kept in contact with
variations over the three years, and it perfectly reflects the value that is wood during the ageing period. A typical example of a phenolic acid that
commonly found in oenological products (Cocchi et al., 2006). is strongly dependent on wood type is gallic acid (Cerezo et al., 2010).
Gallic acid was the most abundant phenolic compound identified,
3.2. Furfurals followed by protocatechuic acid, caffeic and syringic acids and finally p-
coumaric acid. The concentration of the phenolic acids, determined in
When food containing reducing sugars is subjected to heat treatment BVG, agrees well with that reported for wine vinegar and balsamic
under acid conditions, Maillard reactions occur and the formation of vinegar (Cerezo et al., 2008, 2010). From a statistical point of view,
different furan derivatives is observed (Kowalski et al., 2013). Moreover, one-way ANOVA did not show statistically significant differences of the
5-hydroxymethylfurfural and furfural are present in food products that investigated species over the three years, with the only exception of
have been in contact with wood for long periods, for example in aged gallic acid, for which the average concentration of samples of 2015 was
vinegar (Cocchi et al., 2011). As a consequence, the presence of these significantly lower than the average value of 2016, while 2017 samples
species in BVG is not surprising and can be attributed to both the ABM showed an intermediate value (Table 1).
and the concentrated grape must.
As expected, despite showing a high variability, the furfural

Table 1
mean values and associated standard deviation values of the analytes determined in the balsamic vinegar glazes for the three investigated years of production, together
with the one-way ANOVA results (df between = 2; df within = 21). Different superscript letters indicate statistical differences between different years (Tukey multiple
comparison test, P = 95%).
Parameter Units 2015 2016 2017 F p-value*

Glucose g/kg 226a ± 8 223a,b ± 14 211b ± 8 4.793 0.0193

Fructose g/kg 235a ± 10 229a ± 6 216b ± 7 12.121 0.0003
Glu/Fru ratio - 0.96a ± 0.03 0.97a ± 0.06 0.98a ± 0.02 0.507 0.6087
5-HMF mg/kg 956a ± 473 824a ± 591 521a ± 376 1.575 0.2305
Furfural mg/kg 14a ± 12 10a ± 6 7a ± 5 1.494 0.2475
Gallic acid mg/kg 18a ± 6 29b ± 13 23a,b ± 5 3.532 0.0476
Protocatechuic acid mg/kg 12a ± 5 13a ± 4 17a ± 6 1.743 0.1994
Syringic-Caffeic acids mg/kg 4.3a ± 2.3 7a ± 2.9 6a ± 1.8 2.822 0.0821
p-Coumaric acid mg/kg 1.8a ± 0.7 1.3a ± 0.6 1.2a ± 0.4 2.424 0.1129
Dry matter g/kg 594a,b ± 14 601a ± 21 576b ± 8 5.556 0.0116
Ash g/kg 8.2a ± 0.6 8.2a ± 1.1 9.5a ± 2 2.427 0.1127
Total Acidity % 2.1a ± 0.1 2.1a ± 0.1 2.2a ± 0.2 2.422 0.1131
Li mg/kg 0.22a,b ± 0.03 0.25a ± 0.01 0.21b ± 0.02 7.565 0.0034
Na mg/kg 448a ± 30 423a ± 94 451a ± 93 0.306 0.7394
K mg/kg 2872a ± 265 2959a ± 450 3199a ± 723 0.897 0.4229
Mg mg/kg 276a ± 47 248a,b ± 43 213b ± 39 4.111 0.0312
Ca mg/kg 339a,b ± 37 393a ± 68 310b ± 43 5.248 0.0142
Mn μg/kg 1484a ± 496 1857a ± 536 1496a ± 187 1.808 0.1886
Fe mg/kg 7.9a ± 3.3 6.9a ± 2 8.9a ± 2.3 1.069 0.3614
Cu mg/kg 0.47a ± 0.3 0.83a,b ± 0.34 1.01b ± 0.56 3.596 0.0454
Zn mg/kg 1.5a ± 0.9 1.7a ± 0.8 1.2a ± 0.4 0.876 0.4313
Pb μg/kg 64a ± 17 67a ± 28 52a ± 10 1.222 0.3146
*
statistically significant differences (p-value < 0.05) are highlighted in bold.

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L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

3.4. Dry matter is performed for a quality test rather than for nutritional purposes. The
concentration of Li, measured in the analyzed samples, was less than 0.3
Dry matter refers to the material that remains after water removal. mg/kg. One-way ANOVA followed by Tukey test showed a significant
As regards the BVG samples, dry matter is mainly composed of grape difference among samples produced during 2016 and 2017, while 2015
sugars, starch polysaccharides and non-volatile organic acids, in addi­ samples showed intermediate values (Table 1).
tion to minor components, such as inorganic salts and organic com­ As far as Na and K are concerned, the measured concentrations were
pounds at very low concentration that does not affect the measurement quite homogeneous, over the investigated period, also confirmed by
result. The average value of dry matter content, measured on the BVG one-way ANOVA. Overall, Na ranged from 250 to 600 mg/kg, while K,
samples, was, on the whole, about 60% and results agree with those the most abundant cation among those measured, ranged from 2000 to
found for similar products like traditional balsamic vinegars (Masino 4400 mg/kg. Considering that the main ingredient of BVG is concen­
et al., 2008). One-way ANOVA and Tukey test highlighted a statistically trated grape must, the measured values were similar to those determined
significant difference between 2016 and 2017, while the samples in ABTM (Corradini et al., 1996).
collected during 2015 showed intermediate values (Table 1). In partic­ Ca and Mg represent quality parameters for oenological products.
ular, samples produced during 2017 showed a lower dry matter content Generally, Mg/Ca concentration ratio in grape musts and wines is about
than those produced during 2016. This behavior well reflects the sugars 1, even if slightly positive or negative deviations are accepted. This trend
trend and the highlighted difference is probably due to different is also confirmed for the samples under study, where the concentration
composition of the starting raw materials, although changes in the range, for Ca and Mg, was 250-600 mg/kg and 150-350 mg/kg,
production process are also possible. respectively. One-way ANOVA and Tukey test highlighted significant
differences for both the cations in the three years due to a decrease in
3.5. Ash content their content in 2017 compared to the previous years (Table 1).
Concerning heavy metals, Mn, Fe, Cu, Zn and Pb were determined.
Chemical composition of ashes from BVG is due to thermostable The presence of manganese, in oenological products, is directly linked to
compounds, namely metal oxides, phosphates, sulphates, etc. No sta­ the capacity of the vine to absorb this element from the soil; conse­
tistically significant differences were observed among the three years quently, its concentration can be extremely variable depending on the
(Table 1), even if a slightly higher value was observed for 2017 with type of the vineyard soil (Slunjski et al., 2012; OIV, 2018). Mn con­
respect to 2015 and 2016. Even in this case, data results in agreement centration, determined in the BVG samples, ranged from 0.5 to 2.6
with those observed for traditional balsamic vinegars (Masino et al., mg/kg. These values are, on average, lower when compared with those
2008). determined on similar food products (Cocchi et al., 2004).
Iron levels are generally related to machinery used in the production
3.6. Total titratable acidity process such as pressing equipment, which is usually made of iron or
stainless steel. The overall content were in the 4-14 mg/kg range. The
TA represents the sum of the fixed and volatile acids present in a experimental values did not show statistically significant differences in
sample. The TA of the product under investigation is mainly regulated the different years, for both the cations; moreover, as regards the iron
by the amount of ABM used in the formulation since the contribution of content, the measured values were found to be well below that measured
the acidity from the concentrated must (fixed acidity) is quite low. in homologous products, such as ABTM.
As far as vinegars or similar products are concerned, TA is expressed Unlike the previous metals, in wines and musts, the presence of
as the percentage of grams of acetic acid per 100 g of product. This heavy metals such as copper, zinc and lead are subjected to legal limits.
parameter were almost constant for all the investigated samples, with In particular, maximum allowed values are 1, 5 and 0.15 mg/kg for Cu,
annual mean values equal to 2.1 ± 0.1%, 2.1 ± 0.1% and 2.2 ± 0.2% for Zn and Pb, respectively.
2015, 2016 and 2017, respectively. Copper, in the grape juice, originates from soil absorption as for most
Concerning the TA value of the BVG samples, it is not surprising to metals, but the main source is certainly given by copper-based agro­
find such a low acidity value, about 2%, when compared to the mini­ chemical products. The Cu concentration, measured in the BVG samples,
mum acidity value, 6%, required for a PGI ABM. In fact, since BVG is ranged from 0.2 to 1.8 mg/kg, and for 6 samples out of 24, Cu content
obtained by mixing ABM with concentrated grape must, whose acidity is exceeded the fixed limit. Moreover, ANOVA and Tukey test showed
very low, the resulting acidity of the product is expected to be much less significant differences among the years, with Cu levels increasing from
than 6%. 2015 to 2017.
In addition, while the production of ABM is regulated by a specific Concerning zinc, a quite high within-year variability was observed,
production rule that sets a series of physicochemical parameters that and no statistically significant difference was highlighted over the years.
must be strictly complied with, at present, BVG production is not, and Sample concentration ranged between 0.4 and 3.4 mg/kg, lower than
the value of the total acidity has only a technical relevance for the sta­ the fixed limit, for wine and musts, and the concentration values found
bility of the gel conditions of the product. In fact, it is reasonable to in ABTM (Cocchi et al., 2004).
hypothesize that high values of acidity may induce modifications of the As for Zn, the main source of lead, in oenological products, is envi­
starch structure, leading to a possible separation of phases and a ronmental pollution (Angelova et al., 1999). Due to a toxicological
consequent loss of the product rheological characteristics, making it hazard, the maximum acceptable limit of Pb is extremely low. None of
unmarketable. the analyzed samples showed Pb content above the legal limit, with
concentration values ranging from 0.04 to 0.12 mg/kg, and no statisti­
3.7. Metals cally significant differences were observed over the three years.
In summary, the concentration of the heavy metals measured in the
To date, the commercial balsamic reduction is not subjected to any BVG samples was lower than that of homologous products such as
type of restriction as regards the content of metals. Nevertheless, to ABTM. This evidence strongly suggests that the starting raw materials of
evaluate the results obtained in the present study, the metal concen­ BVG were previously subjected to processes for the reduction of the
tration limits, fixed for wines and musts (OIV, 2017) and the literature metal content through demetallization practices.
data concerning homologous products such as ABTM (Corradini et al.,
1996, Cocchi et al., 2004), were taken into consideration. 3.8. Exploratory data analysis
Concerning Li, the natural content of this metal in wines is very low
(Catarino et al., 2006; Gomez et al., 2004); therefore, its determination Principal Component Analysis was applied to the whole dataset, with

5
L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

size {24 × 22}, including the values of the 22 physicochemical param­

eters listed in Table 1 for the 24 BVG samples considered in this work,
ordered according to the date of production. Based on the scree plot, two
principal components (PCs) were selected, accounting for 40.11 % of
total data variance. No outliers were detected based on the inspection of
the Q residuals vs. Hotelling T2 plot (Fig. 2), suggesting that overall the
composition of the analyzed batches is quite constant.
The PC1-PC2 score plot (Fig. 3) highlights that the samples of 2015
and 2016 are quite overlapped with each other and that samples of 2015
are separated in two clusters. In particular, the cluster including samples
n. 4, 5 and 6, corresponding to the batches produced in mid-2015, lies at
high positive values of PC2, due to the relatively high values of glucose,
fructose and dry matter (Fig. 4).
A clear difference was instead observed between the BVG samples
produced in 2017 and those of the previous two years. Except for sample
n. 18, which corresponds to the first analyzed batch of 2017, the
remaining 2017 samples are found on the lower part of the PC1-PC2
graph, together with sample 17, which corresponds to the last
Fig. 3. PC1 vs. PC2 scores plot.
analyzed batch of 2016. These samples show tendentially high values of
total acidity, Na, Fe and Cu, and low values of fructose, glucose, dry
matter, Li, Mg and Ca (Fig. 4). This trend is coherent with the results of
one-way ANOVA and Tukey multiple comparison test, except for total
acidity, Na and Fe, which did not show statistically significant differ­
ences among the three years, mainly due to sample 18. The difference
between the samples produced in 2017 and those of 2015 and 2016, is
probably due to the fact that the formulation of BVG was changed to
meet, on the one hand, a higher technological stability of the product
and, on the other hand, consumers’ preferences.

4. CONCLUSIONS

Balsamic vinegar glaze is a product obtained by mixing concentrated

grape must and ABM, and addition of a small amount of cornstarch in
order to obtain a creamy consistency. Often consumers choose BVG as an
alternative to the most expensive ABTM, whose production is regulated
by a precise and specific production rule (Gazzetta Ufficiale, 1987), as
for ABM. Contrary, to date, BVG is not subjected to any regulation that
limit, for example, the quantity or the type of the raw material used in
Fig. 4. PC1 vs. PC2 loadings plot.
the formulation of BVG. Moreover, to the best of our knowledge, to date,
scientific literature reports no studies about BVG.
As a consequence, the present investigation is a preliminary study of procedures for process-control purposes and hazards control, based on
the chemical characterization, in terms of main components and bulk objective variables typical of the product and, indirectly, of some of its
parameters, of the BVG. Investigated variables helped improving main raw materials.
knowledge about product characteristics, in terms of composition. The To this end, 24 samples of BVG, representative of three years of
present research may constitute a starting point to develop monitoring production were sampled from a local producer. Results obtained from
the analysis of the samples of cream showed that the production process
is well controlled as chemical parameters were mainly unchanged dur­
ing the investigated period. During the considered production years, the
balsamic glaze composition slightly changed towards to higher acidity
values and lower sugar content.
The marketable product preserves the chemical characteristics of the
starting raw materials such as ABM, although showing significant vari­
ations of some parameters such as glucose, fructose and total titratable
acidity.
Concerning metals determination, the low content of heavy metals,
detected in the samples, probably implies a preventive demetallization
of the starting raw materials, despite some exceptions being observed.
As for the content of metals, i.e. Cu, Zn and Pb, for which there is a legal
limit for wine and grape musts, the analyzed BVG samples showed
modest values, which guarantee the law requirements.
It should be taken into due consideration, however, that the formu­
lation of BVG consists of a mixture of raw materials coming from supply
chains with different product characteristics. Therefore, the final con­
tent of the chemical species is the result of a diversified and weighted
Fig. 2. Plot of the Hotelling T2 values vs. Q residuals for the calculated contribution of products with an intrinsic variability. Starting from these
PCA model. results, further research work will be focused on an extensive

6
L. Lancellotti et al. Journal of Food Composition and Analysis 94 (2020) 103620

characterization of BVG samples coming from different producers of the Consonni, R., Cagliani, L.R., Rinaldini, S., Incerti, A., 2008. Analytical method for
authentication of Traditional Balsamic Vinegar of Modena. Talanta 75, 765–769.
same geographic area.
Corradini, F., Franchini, G., Marchetti, A., Preti, C., 1996. Determination of alkali and
alkaline earth metals in Aceto Balsamico Tradizionale di Modena by flame atomic
Declaration of Competing Interest absorption spectroscopy. Journal of AOAC International 79, 1167–1173.
Gazzetta Ufficiale della Repubblica Italiana 1987 No 44 del 23/02/1987. D.M: 9/02/
1987.
The authors declare that they have no known competing financial Glatt, H., Sommer, Y., 2006. 15 - Health risks of 5-hydroxymethylfurfural (HMF) and
interests or personal relationships that could have appeared to influence related compounds. In: Skog, K., Alexander, J. (Eds.), Woodhead Publishing Series in
the work reported in this paper. Food Science, Technology and Nutrition. Acrylamide and Other Hazardous
Compounds in Heat-Treated Foods. Woodhead Publishing, pp. 328–357. https://doi.
org/10.1533/9781845692018.2.328. ISBN 9781845690113.
Funding Gomez, M.D.M.C., Brandt, R., Jakubowski, N., Andrersson, J.T., 2004. Changes of the
metal composition in German white wines through the winemaking process. A study
of 63 elements by inductively coupled plasma -mass spectrometry. Journal of
This research did not receive any specific grant from funding Agricultural and Food Chemistry 52, 2953–2961.
agencies in the public, commercial, or not-for-profit sectors. Kowalski, S., Duda-Chodak, A., Lukasiewicz, M., Ziec, G., 2013. 5-Hydroxymethyl-2-
furfural (HMF) – Heat-induced formation, occurrence in food and biotransformation
– a Review. Polish. Journal of Food and Nutrition Sciences 63 (4), 207–225.
CRediT authorship contribution statement Kumari, J.D., 2012. Health benefits of polyphenols and prevention of diseases in humans.
International Journal of Pharmaceutical Research and Bio-Science 1 (6), 166–189.
Lisa Lancellotti: Investigation. Alessandro Ulrici: Formal analysis, Lalou, S., Hatzidimitriou, E., Papadopoulou, M., Kontogianni, V.G., Tsiafoulis, C.G.,
Gerothanassis, I.P., Tsimidou, M.Z., 2015. Beyond traditional balsamic vinegar:
Writing - review & editing. Simona Sighinolfi: Methodology, Formal
Compositional and sensorial characteristics of industrial balsamic vinegars and
analysis, Writing - original draft. Andrea Marchetti: Conceptualization, regulatory requirements. Journal of Food Composition and Analysis 43, 175–184.
Project administration, Writing - review & editing. Lemmetti, F., Giudici, P., 2011. Simple chemical and physycal parameters to identify
aged balsamic vinegars. Industrie delle bevande 40, 12–23.
Manzini, S., Durante, C., Baschieri, C., Cocchi, M., Sighinolfi, S., Totaro, S., Marchetti, A.,
Appendix A. Supplementary data 2011. Optimization of a dynamic headspace-thermal desorption-gas
chromatography/mass spectrometry procedure for the determination of furfurals in
Supplementary material related to this article can be found, in the vinegars. Talanta 85, 863–869.
Masino, F., Chinnici, F., Bendini, A., Montevecchi, G., Antonelli, A., 2008. A study on
online version, at doi:https://doi.org/10.1016/j.jfca.2020.103620. relationships among chemical, physical, and qualitative assessment in traditional
balsamic vinegar. Food chemistry 106, 90–95.
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