Article

Quality characteristics of wheat flour dough and

bread containing grape pomace flour

Monika Šporin1, Martina Avbelj2, Boris Kovač1,3 and

Sonja Smole Možina2

Abstract
Wheat bread was enriched with 6%, 10% and 15% dried and milled grape pomace flour from two grape
cultivars: ‘Merlot’ and ‘Zelen’. Rheological, textural, sensory and antioxidant properties of the enriched dough
and bread were evaluated, and compared to control samples. Grape cultivar had significant impact on the
rheological characteristics of the dough, and on the sensory and antioxidant properties of the final bread.
Development time and dough stability were longer when ‘Merlot’ grape pomace flour was added compared to
‘Zelen’ grape pomace flour and the control. Grape pomace flour addition affected bread volume, firmness,
crumb and crust colour, and odour and taste intensity. Moreover, grape pomace flour addition resulted in a
stickier and less springy crumb texture, and some negative sensorial properties, such as increased intensity
of aftertaste and sand feeling in the mouth. The phenolic content and antioxidant activity of bread were
positively correlated with grape pomace flour addition (r ¼ 0.987, p ¼ 0.01 and r ¼ 0.941, p ¼ 0.01 between
phenolic content and ferric reducing antioxidant power and phenolic content and 2,2-diphenyl-1-picrylhydra-
zyl, respectively). The highest total phenolic contents were 5.92 mg gallic acid equivalents (GAE)/g dw for
‘Merlot’ and 3.65 mg gallic acid equivalents /g dw for ‘Zelen’, which were seen for the bread prepared with the
highest grape pomace flour addition (15%). The highest antioxidant activity determined by the 2,2-diphenyl-1-
picrylhydrazyl and ferric reducing antioxidant power assays were seen for the bread prepared with the highest
‘Merlot’ grape pomace flour addition (15%). Dough characteristic and sensory profile are strongly influenced
by cultivar of grape pomace flour. Based on results of sensory profiling, the variety ‘Zelen’ is suggested
for use.

Keywords
Grape pomace, bread, sensory properties, technological properties, antioxidant properties
Date received: 30 June 2017; accepted: 7 November 2017

Agro-industrial wastes that originate from food pro-

INTRODUCTION
Bakery products are widely consumed as staple foods
all over the world. Due to their importance in the diet
and the low costs of their ingredients, bakery products
can be supplemented with components of high nutri-
tional value. The concept of functional foods and the
importance of the relationships between foods and
health have increased the impact on food innovation.
Food Science and Technology International 0(0) 1–13
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DOI: 10.1177/1082013217745398
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duction can become ingredients of added value due to
their potential phytochemical contents, with the asso-
ciated significant health benefits.
Grapes (Vitis vinifera) are one of the largest fruit
crops throughout the world, and they are mainly used
for wine production. Europe produced over 28,000,000
1
Mlinotest Živilska Industrija d.d., Ajdovščina, Slovenia
2
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
3
Faculty of Health Sciences, University of Primorska, Izola,
Slovenia
Corresponding author:
Monika Šporin, Mlinotest Živilska Industrija d.d., Tovarniška cesta
14, 5270 Ajdovščina, Slovenia.
Email: monika.sporin@mlinotest.si
Food Science and Technology International 0(0)
tonnes of grapes in 2013 (Food and Agriculture
Organization of the United Nations, 2015). It is esti-
mated that after grape juice pressing, around 20% of
the total weight of grapes used for wine remains as a
solid residue, known as the grape pomace (Brenes et al.,
2016). Large amounts of such by-products can repre-
sent serious environmental and disposal problems for
wineries. On the other hand, grape pomace represents
an excellent source of various ‘high-value’ molecules,
such as flavanols, flavonols, catechins, anthocyanins,
stilbenes and phenolic acids, and of dietary fibre
(Devesa-Rey et al., 2011; Katalinić et al., 2010; Trošt
et al., 2016). Indeed, numerous studies have indicated
that grape polyphenols are associated with prevention
or delay of chronic degenerative diseases, such as ath-
erosclerosis, cancer, cardiovascular disease and type 2
diabetes (Ferri et al., 2016; Jara-Palacios et al., 2015;
Kadouh et al., 2016; Nunes et al., 2016).
A few studies have reported the addition of grape
pomace to foods. The reasons behind the addition of
grape pomace to different foods can be varied, from
improving the sensory properties to increasing the
nutritional value. Riazi et al. (2016) used different
levels of dried and ground red grape pomace to study
the oxidation and colour properties of nitrite-reduced
meat emulsion systems. Pedroza et al. (2013) added
dehydrated waste grape skins from the juice industry
to aged and young red wines as an innovative way to
counteract the colour loss before bottling. Lavelli
et al. (2014) used dried and ground grape skins for
the development of a new tomato-based products.
They reported that the reducing capacity was much
higher in all of their fortified tomato purees than in
their controls. Marchiani et al. (2016) used grape
skin flours as sources of polyphenolic compounds in
yoghurt formulation. Yoghurt that contained grape
skin flours showed significantly higher total phenolics
content and antioxidant activity. Grape pomace
powder has been used as a source of antioxidant dietary
fibre in yoghurt salad dressings (Tseng and Zhao,
2013). Özvural and Vural (2011) incorporated grape
seed flour into frankfurters to improve the nutritional
profile and reduce lipid oxidation. Sant’Anna et al.
(2014) incorporated grape pomace powder into fettuc-
cini (ribbon-like) pasta to improve the nutritional
value. This increased the polyphenol content, but
had a negative impact on the sensory properties.
Incorporation of defatted grape seed powder at 5%
decreased free fatty acid formation in biscuits
(Aksoylu et al., 2015). Pasqualone et al. (2013, 2014)
produced and characterized biscuits supplemented with
grape pomace extract (cv. Sangiovese) and, while
improving the nutritional properties, they observed
higher phenolic contents and antioxidant activity.
2
Mildner-Szkudlarz et al. (2011) used milled red
grape by-products as a source of dietary fibre and phen-
olic compounds in rye sourdough bread, although
they did not indicate the grape variety. Hoye and
Ross (2011) tested the impact of frozen storage on con-
sumer acceptance and total phenolic content (TPC) of
bread made with varying levels of grape seed flour.
Some studies have also tested supplementation of
bread by previously prepared grape seed extracts,
which increased the antioxidant capacity of the bread
(Peng et al., 2010). Thus the addition of grape by-
products to bread has resulted in increased antioxidant
value and changed sensory properties in previous
studies.
Based on these previous studies, we can conclude
that grape pomace should be an excellent raw material
for nutritional enrichment of bread. However, previous
studies did not answer many further important aspects.
Some studies focused on grape seeds and extracts, with-
out using grape skins, and there was no indication of
the grape variety in the study on rye bread (Mildner-
Szkudlarz et al., 2011). The aim of the present study
was to compare the effects of grape pomace flour (GPF)
from red ‘Merlot’ grapes and white ‘Zelen’ grapes on
the rheological, textural, sensory and antioxidant prop-
erties of wheat bread. Furthermore, the initial grape
substrate was minimally processed, with only drying
and milling used.
MATERIALS AND METHODS
Materials
The ‘Merlot’ (red) and ‘Zelen’ (white) grape (Vitis vini-
fera) pomaces originated from a local producer in the
Vipava area in south-western Slovenia, with the grapes
harvested in 2013. Wheat flour (Triticum aestivum)
(9.9% protein, 13.08% moisture, 0.49% ash) was pro-
vided by Mlinotest (Slovenia), yeast (Saccharomyces
cerevisiae, Digo) was provided by Kvasac (Croatia),
and enzymatic improver (Oxigen; Ingredients: wheat
flour, enzymes: alpha amylase, amyloglucosidase, glu-
cose-oxidase, xylanase) was provided by Lesaffre
(France).
Chemicals
Ethanol (96%) was from Itrij (Kropa, Slovenia),
sodium carbonate and gallic acid were from Merck
(Darmstadt, Germany), and methanol (99.9%),
2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent, Folin-
Ciocalteu reagent and Trolox were from Sigma-
Aldrich GmbH (Steinheim, Germany). All reagents
were of analytical quality. Ultrapure water was used
(MilliQ, Millipore) for preparation of solutions.
 Šporin et al.

Sample preparation
Bread preparation. The grape pomace was from the
skins and seeds of the grapes, and it was stored at
20  C until it was dried for 3 h at 80  C in a convection
oven (Dibas, Wiesheu, Germany). The dried pomace
was milled to flour using an AR100 grinder
(Moulinex, France), and passed through a 250-mm
sieve. The nutritional composition of the ‘Zelen’ and
‘Merlot’ GPF is given in Table 1. The wheat flour
was enriched with this GPF at 0% (no addition con-
trol), 6%, 10%, and 15% (w/w). For dough prepar-
ation, 2% yeast, 2% salt, 1% enzymatic improver
and 65% water were used (all w/w). All the ingredients
were mixed in a spiral mixer (IP10F; Fimar, Italy) at
low speed for 4 min, followed by high speed for 6 min.
Once mixed, the dough was left to rest for 15 min at
22  C. The dough was then divided into 440 -g pieces,
moulded manually, and placed into baking pans. After
fermentation at 30  C and 85% relative humidity for
60 min, the loaves were baked in a convection oven
(Dibas, Wiesheu, Germany) at 200  C for 10 min, fol-
lowed by 180  C for 11 min.
GPF composition determination
Protein (Nx6,25), fat and moisture were determined
according to ISO 16634-1:2008, 1444:1973, 1442:1997,
respectively; Total dietary fibre were determined fol-
lowing 985.29 AOAC method; Soluble dietary fibres
were determined following 993.13 and insoluble
991.42 AOAC methods; Total carbohydrates were cal-
culated by difference (g) ¼ 100 -g crude (water þ ash þ
fats þ proteins þ fibre).
Sample extraction. The breads were sliced and dried in
an electric oven at 50  C for 12 h, and then ground with
an electric grinder (AR100 grinder, Moulinex, France).
Three grams of sample (bread, wheat flour, GPF) were
extracted with 25 mL 70% ethanol at 30  C for 20 min
in an ultrasonic water bath (Sonis10; Iskrapib). This
mixture was then centrifuged at 9000  g for 5 min
(3K30; Sigma), and the supernatant was filtered
through Whatman No. 1 filter paper. The extraction
step with 25 mL 70% ethanol was repeated three
times. All three filtered fractions were pooled as the
crude extracts.
Solid phase extraction of phenolic compounds. A
reverse phase column (Strata-X, catalogue number:
8B-S100-EBJ; Phenomenex, US) was placed on the
vacuum system and first conditioned with 3 mL 100%
methanol, followed by equilibration with 3 mL 10%
aqueous methanol. The crude extract (5 mL) was ini-
tially diluted with 20 mL water, and then applied to the
column. The columns were then washed with 3 mL 10%
aqueous methanol, and dried using the vacuum pump.
All of the bound compounds were subsequently eluted
with 3 mL methanol. Finally, the solvent was evapo-
rated and the samples were filtered through 0.2 -mm
filters and resuspended in 1 mL 50% methanol.
Measurement of quality characteristics
Dough characteristics. The effects of the GPF on
dough rheology during mixing were determined using a
farinograph (300 -g flour capacity; Brabender, Duisburg,
Germany), following the American Association of
Cereal Chemists (AACC) International (2000) method
54-29. The farinograph analysis was conducted on the
samples of wheat flour blended with the GPF levels of
0 g/100 g, 6 g/100 g, 10 g/100 g and 15 g/100 g flour (i.e.
0%, 6%, 10%, 15% GPF). The farinograph parameters
are expressed as water absorption (%), dough develop-
ment time (min), stability (min) and degree of softening
(BU), and farinograph quality number (mm).

Volume and specific volume. The bread was weighed

Table 1. Nutritional composition of ‘Merlot’ and ‘Zelen’ after cooling and the volume (mL) was determined by
grape pomace flour the seed displacement method, according to AACC
‘Merlot’ grape ‘Zelen’ grape International (2000) method 10-05.01. The specific
Content per 100 g pomace flour pomace flour volume (mL/g) was calculated as the ratio of the loaf
volume to the bread weight.
Total fat (g) 13.9  0.6 8.5  0.4
Total carbohydrates (g) 12.2  2.4 19.7  2.5 Bread firmness. The bread firmness was measured
Sugars (g) 1.66  0.09 13.71  0.73 using a texture analyser (TA-XT Plus; Stable
Total dietary fibre (g) 54.5  2.3 50.3  2.2 Micro Systems, UK) equipped with an aluminium 35-
Soluble (g) 3.0  0.6 5.3  0.7 mm-diameter cylindrical probe, according to AACC
Insoluble (g) 51.1  2.2 44.3  2.4 International method 74-09 (Bourne, 1995). Two
Protein (g) 11.3  0.3 10.6  0.3 slices of 12.5-cm thickness were compressed to 40%
Moisture (g) 3.9  0.3 6.5  0.3 of the original height at a crosshead speed of 10 mm/
s. The resulting peak force of compression was reported
Ash (g) 4.20  0.16 4.36  0.17
as the bread firmness.

3
Food Science and Technology International 0(0)
Bread colour. The colour of the bread slices was mea-
sured using a colorimeter (Minolta CR 200; Konica
Minolta, Osaka, Japan). The data are expressed as
L*, a* and b*, where L* indicates whiteness (100) or
blackness (0), a* indicates red (positive) or green
(negative) and b* indicates yellow (positive) or blue
(negative). The colorimeter was calibrated using a
standard. Results are given as the colour differential
(E) between control bread and bread with added
‘Zelen’ and ‘Merlot’ GPF, calculated as follows
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
E ¼ L2 þ a2 þ b2
where L is calculated as L*control bread  L*bread with
GPF, a is calculated as a*control bread  a*bread with GPF,
and b is calculated as b*control bread  b*bread with GPF
(Sant’Anna et al., 2014).
Sensory profiling
A sensory panel that consisted of eight trained experts
(five females, three males; age range, 28–54 years) was
used to evaluate the bread samples. All of the experts
had experience in descriptive evaluation of bread. The
initial panel discussion and training session was com-
pressed to a total of two 3-h sessions. A sensory profile
that included the attributes related to texture, aroma,
flavour/taste and ‘mouth feel’ was completed according
to the ISO 13299:2003 standard (2003), in the sensory
laboratory of Mlinotest Food Industry. In the first
phase of the test, the panel developed 25 descriptors
for the sensory attributes of the control bread and the
bread with GPF addition. The panellists created a
10-point unstructured line scale with descriptor labels
at either end. The control bread was chosen as the ref-
erence sample, to reduce the variation among the pan-
ellists. The panellists defined the intensity value of the
reference sample with respect to each sensory attribute.
In the second phase of the test, breads with GPF add-
ition were evaluated in comparison to the control
bread. The panellists rated the samples (one whole
loaf of bread, two slices/person) individually, on the
basis of the questionnaires, using a balanced test
design in which the serving order was randomised for
each panellist. The scaled responses were converted to
numeric values, from 0 to 10 (Bagdi et al., 2016).
Estimation of total polyphenol content
TPC of the GPF, wheat flour and bread extracts were
determined spectrophotometrically using Folin-
Ciocalteu reagent (Singleton et al., 1999). A 1:10 dilu-
tion of Folin-Ciocalteu reagent and 7.5% Na2CO3 were
prepared. Then 20 mL of each extract obtained with
4
solid phase extraction was put into a microtitre
plate, followed by addition of 100 mL diluted Folin-
Ciocalteau reagent and 80 mL sodium carbonate
solution. After a 2-h incubation in the dark, the absorb-
ances were measured at 760 nm using a microplate
reader (Safire2; Tecan, Austria). Gallic acid was used
as the reference standard, and the data are expressed as
gallic acid equivalents (GAE), as mg GAE/g sample, on
a dry mass basis.
Determination of antioxidant properties
ð1Þ
DPPH assay. Radical scavenging activities were deter-
mined by the method of Brand-Williams et al. (1995),
using DPPH as the free radical. In this assay, the purple
chromogen radical is reduced by antioxidant/ reducing
compounds to the corresponding pale yellow hydra-
zine, and this change can be monitored and quantified
using a spectrophotometer at 515 nm to 528 nm
(Karadag et al., 2009). Due to the large number of
samples, a microtitre plate was used and the absorb-
ance was measured with a microplate reader. Here,
25 mL sample obtained from the solid phase extraction
was diluted with 25 mL distilled water and added to
1 mL 0.05 mg/L DPPH solution, and mixed by vortex-
ing. The mixture was incubated in the dark at room
temperature for 30 min (Ho et al., 2013). After incuba-
tion, 200 mL of the solution was transferred to a trans-
parent microtitre plate. The absorbance was measured
at 517 nm with a microplate reader. The data are
expressed as GAE, as mg GAE/g sample, on a dry
mass basis.
FRAP assay. The ferric reducing antioxidant power
(FRAP) assay is based on the reduction of yellow
ferric–tripyridyltriazine complex (Fe(III)–TPTZ) to
blue ferrous complex (Fe(II)–TPTZ) by the phenolics,
through the action of electron-donating antioxidants
(Benzie et al., 1999). The FRAP solution consisted of
300 mM acetate buffer, pH 3.6, 10 mM 2,4,6-tripyridyl-
s-triazine, in 40 mM HCl and 20 mM FeCl3  6H2O
(10:1:1, v/v/v). Then 20 mL of the extract obtained
from the solid phase extraction was mixed with
280 mL FRAP solution, followed by a 30 min incuba-
tion in the dark. The absorbance was measured at
593 nm with a microplate reader. The data are
expressed as mg Trolox equivalents (TE)/100 g
sample, on a dry mass basis.
Statistical analysis
The data are presented as means  standard deviation,
for three replicates. Statistical analysis was conducted
using one-way analysis of variance (ANOVA), followed
by two post-hoc tests to determine any differences
 Šporin et al.

between group means: where equal variances were
assumed, Tukey’s multiple range tests; and where
non-equal variances were assumed, Games-Howell
tests. For the sensory parameters, the median values
were calculated. Possible differences between the vari-
ous breads with respect to the sensory parameters were
analysed using Friedman tests, followed by Wilcoxon
signed rank tests. Differences with p < 0.05 were con-
sidered to be statistically significant. Pearson correl-
ation coefficients were calculated for pairs of
biochemical parameters and dough characteristics.
Statistical analyses were conducted using SPSS 22.0
software (SPSS Inc., Chicago, IL).
RESULTS AND DISCUSSION
Dough characteristics
The addition of GPF significantly affected the dough
mixing according to the farinograph. The farinograph
parameters are expressed as water absorption, dough
development time, stability and degree of softening,
and farinograph quality number (Table 2). GPF
addition significantly affected water absorption. For
the dough with the ‘Merlot’ GPF addition (i.e. the
‘Merlot’ GPF dough), at all levels, the water absorption
was lower than for the control bread. The data
obtained are contrary to other studies, where the add-
ition of fibre or extracts resulted in greater water
absorption (Boubaker et al., 2016; Huang et al., 2016;
Park et al., 2012). Gluten and some polysaccharides
naturally present in wheat flour highly contribute to
better water absorption of dough. A lack of such poly-
saccharides in the ‘Merlot’ GPF dough, which are
responsible for water absorption, and the dilution of
the gluten due to GPF addition, might have contribu-
ted to this lower water absorption. On the contrary, the
‘Zelen’ cultivar did not cause significant variations of
dough water absorption, except at 10% level, that
induced a significant increase. This result points out a
relation between grape cultivar (i.e. grape pomace com-
position in terms of main and minor compounds, or
even enzymes) and dough properties.
The dough development time and its stability are
indicators of the flour strength, where higher values
indicate stronger doughs. Compared to the control,
‘Zelen’ GPF dough showed no statistically significant
change in development times, while for the ‘Merlot’
GPF dough the development time was longer for all
of the tested GPF additions. ‘Merlot’ dough, with
15% GPF additions showed the greatest impact on
development time.
The addition of ‘Zelen’ (6%) and ‘Merlot’ (10%,
15%) GPF to the dough increased its stability time
compared to the control, which indicates improved
dough strength and bread-making ability. The decrease
in the wheat protein caused by the GPF additions
would normally result in decreased dough strength.
However, for the ‘Merlot’ GPF dough, the strength
might have been improved by the phenolic substances
in the GPF. There are positive correlations between
TPC and stability time for ‘Merlot’ dough (r ¼ 0.970;
p < 0.01). This phenomenon has already been reported
by Anil (2017). The addition of the ‘Zelen’ GPF to the
dough had an opposite effect, with a decrease in dough
stability. The ‘Merlot’ GPF dough also showed
decreased degree of softening, which can be explained
also by the higher levels of phenolic substances in the
dough (r ¼ 0.941; p < 0.01). However, for the ‘Zelen’
GPF dough, the opposite occurred. The reason for
the increase in the softening degree might relate to the
reduced gluten content and the destruction of the
gluten network (Nikolić et al., 2013).
The farinograph quality number is defined as the
length from the water point to a point 30 farinograph

Table 2. Farinograph parameters for wheat flour dough (control) and doughs with ‘Merlot’ or ‘Zelen’ grape pomace flour
additions (6%, 10%, 15%).a

Farinograph dough parameters

Addition Water absorption Development Stability Degree of Quality

Condition (%) (%) time (min) (min) softening (BU) number (mm)

Control None 58.47  0.15a 2.0  0.12a 12.14  0.49a 20.3  3.2a 122.3  10.0a
‘Merlot’ grape 6 57.83  0.06b,d 6.22  0.13b 11.01  0.07a 22.0  2.0a 108.3  1.5a
pomace flour 10 57.53  0.06b 6.41  0.22b 15.20  0.13a,c 14.3  0.6a 130.3  2.3a
15 57.17  0.15b 9.51  0.27c 25.28  0.29b 2.6  1.5b 221.7  0.6b
‘Zelen’ grape 6 58.60  0.26a 1.41  0.06a 16.10  1.01a,c 21.0  4.6a 129.7  13.0a
pomace flour 10 59.10  0.00c 1.32  0.03a 5.39  0.37d 43.3  2.1c 33.0  0c
15 58.17  0.15a,b,d 1.22  0.02a 4.21  0.15d 84.0  4.0d 45.7  2.9c
a
Data are means  standard deviation (n ¼ 3). Data with different alphabets in the same column are significantly different (p < 0.05).

5
Food Science and Technology International 0(0)
2000
a a,b
1800
a b
1600
a c
1400
a
1200
Firmness (N)
Volume (mL)
b 2
b,c
1000
800
600
400
200
0 0
Control M6 M10
Firmness (N)
Figure 1. Volume, specific volume and firmness of bread prepared from wheat flour dough (control) and doughs with the
additions of 6%, 10% and 15% ‘Merlot’ grape pomace flour (M6, M10, M15, respectively) or ‘Zelen’ grape pomace flour
(Z6, Z10, Z15, respectively). Data are means  standard deviation (n ¼ 3). Columns with different alphabets within the
same measure are significantly different (p < 0.05).
units (FU) below the centre line of the greatest consist-
ency along the time axis (Yanga et al., 2014). The add-
ition of the ‘Merlot’ and ‘Zelen’ GPFs to the dough
influenced the farinograph quality number. For the
‘Zelen’ GPF dough, the farinograph quality numbers
were the same as (at 6%) or significantly lower than
(at 10%, 15%) the control dough. Conversely, the far-
inograph quality number of the ‘Merlot’ GPF dough
was the same as or higher than the control dough.
Volume and firmness
The volume, specific volume and firmness data are illu-
strated in Figure 1, which illustrates the significant dif-
ferences for the volumes and specific volumes between
the control bread and the GPF dough breads. The 15%
‘Zelen’ GPF addition resulted in the lowest bread
volume, on the other hand there were no significant
differences in bread volume between the various
‘Merlot’ GPF additions. Addition of 6% GPF had
the lowest impact on bread volume.
Bread firmness is commonly used as an indicator of
bread quality. In the present study, when compared to
the control, only the additions of 15% ‘Merlot’ and 6%
‘Zelen’ GPF had significant impact on crumb firmness
as shown in Figure 1.
The data agree with previous findings, where breads
with any gluten-free source resulted in smaller volumes
6
3.5
ab a,b
b 3
b,c
c a,b
d 2.5
Specific volume (mL/g)
d b,c
b,c b,c
d
1.5
1
0.5
M15 Z6 Z10 Z15
Volume (mL) Specific volume (mL/g)
and specific volumes compared to the control breads
(Frutos et al., 2008; Hoye and Ross, 2011; Jensen
et al., 2015). Previous studies have shown that bread
firmness increases with grape seed flour addition (Hoye
and Ross, 2011). However, fortification with grape seed
extract had no influence on the firmness of the bread
(Peng et al., 2010). A significant reduction in specific
bread volume and increase in firmness has also been
reported in similar studies where bread was enriched
by lemon fibre (Fu et al., 2015), turmeric powder
(Lim et al., 2011), quinoa leaves (Świeca et al., 2014)
and ground flaxseed hulls (Seczyk et al., 2017).
The reduction of bread volume might also be
explained by the enzyme and yeast activities.
Amylases in the enriched dough might be inhibited by
the phenolic compounds in the GPFs, which would
result in inadequate levels of maltose. This might also
result in limited gas production by the yeast, which
would subsequently lead to bread with smaller
volume and relatively compact texture. Similar effects
were noted by Wang et al. (2007) and Zhang and
Kashket (1998).
Some dough characteristics were correlated with the
volume of the final product. The volume of final bread
was in positive correlation (r ¼ 0.875; p ¼ 0.01) with
‘Zelen’ GPF dough but in negative correlation
(r ¼ 0.809; p ¼ 0.01) with ‘Merlot’ GPF dough devel-
opment time. The greatest differences in volume of the
 Šporin et al.

control bread and the GPF dough breads were with
10% and 15% GPF additions. When ‘Zelen’ GPF
was added it resulted in positive correlation between
volume and dough stability (r ¼ 0.814; p ¼ 0.01). The
volume of final bread was in negative correlation
(r ¼ 0.962; p ¼ 0.01) with ‘Zelen’ GPF dough degree
of softening. The degree of softening was the highest
and the volume was the lowest when 15% of ‘Zelen’
GPF was added.
Bread colour
The GPF enrichment had a significant impact on the
bread crumb and crust colours, with the results given in
Table 3 and illustrated in Figure 2.
The bread with the GPF additions showed decreased
L* and increased a* values. Thus, ‘Merlot’ GPF enrich-
ment resulted in decreased brightness and increased
dark rose colour of the crumb, while ‘Zelen’ GPF add-
ition resulted in lower a* values. ‘Merlot’ GPF also
resulted in decreased b* values, which indicates the
presence of a blue colour, while ‘Zelen’ GPF resulted
in increased b* values, thus indicating a yellow colour.
Anthocyanins are the most abundant polyphenolic
compounds in coloured grapes (Ferreira et al., 2016)
and they contribute to the red, purple and blue pigmen-
tation of ‘Merlot’ GPF enriched bread. Flavonols are
yellow pigments that directly contribute to the colour
of white wines (Castillo-Munõz et al., 2010), and con-
sequently also to the more yellow colour of ‘Zelen’

Table 3. Colour of the bread slices prepared from the wheat flour dough (control) and doughs with ‘Merlot’ or ‘Zelen’
grape pomace flour additions (6%, 10%, 15%).a

Colorimeter data
Addition
Condition (%) L* a* b* E
–
Control None 72.67  1.65a  0.99  0.11a 13.27  0.60a
‘Merlot’ grape 6 46.88  2.16b 6.42  0.25b 8.86  0.49b 27.3  2.5a
pomace flour 10 43.35  1.60c 7.93  0.28c 8.37  0.44c 31.0  2.2b
15 38.76  2.25d 9.22  0.42d 8.04  0.66d 35.7  2.8c
‘Zelen’ grape 6 51.44  2.04e 3.98  0.38e 13.11  0.58a 21.0  2.4d
pomace flour 10 48.65  1.97f 5.05  0.46f 14.88  0.52e 24.1  2.5e
15 45.43  1.27g 5.71  0.35g 15.37  0.43f 28.2  1.9a
a
L* indicates whiteness (100) or blackness (0), a* indicates red (positive) or green (negative), b* indicates yellow (positive) or blue
(negative) and E total colour difference. Data are means  standard deviation (n ¼ 3). Data with different alphabets in the same column
are significantly different (p < 0.05).

Figure 2. Slices of the bread prepared from wheat flour dough (control) and doughs with additions of 6%, 10% and 15%
‘Merlot’ grape pomace flour (a: M6, M10, M15, respectively), and additions of 6%, 10% and 15% ‘Zelen’ grape pomace
flour (b: Z6, Z10, Z15, respectively).

7
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Table 4. Attributes and descriptive terms of the sensory profile of the bread prepared from the wheat flour dough (control) and doughs with ‘Merlot’ or ‘Zelen’ grape
pomace flour additions (6%, 10%, 15%).a

Crust appearance Bread slice properties Bread texture

Homogeneity Crumb pores

Add. Crust Crumb Sand
Condition (%) Colour Surface Colour thickness Homogeneity Size colour Cohesiveness Springiness Toughness Hardness Crumbliness Adhesivity feeling

Control None 7.5a 8.5a 2.0a 5.0a 8.0a 5.0a 1.0a 0.0a 10.0a 8.0a 6.0a 0.0a 3.0a 0.0a
‘Merlot’ 6 8.25a,b 8.75a 5.0b 5.0a 5.0b 6.25b 6.25b 2.0b 8.0b 7.0b 5.0b 1.5b 3.25a 3.0b
Food Science and Technology International 0(0)

grape
Pomace 10 8.75b 8.0b 6.5c 5.0a 6.0c 5.75a,b 8.0c 1.0b 8.5b 6.0b 5.0b 2.5c 4.0b 4.0c
flour
15 8.5b 7.75b 8.0c 5.5a 6.0c,d 6.0a,b 9.0b 1.0b 7.0b 6.0b 4.75b 3.0c 4.0b 6.0d
‘Zelen’ 6 8.5a,b 8.0a,b 4.25d 5.0a 6.75c 6.0a,b 4.75e 0.0a 9.0b 7.0b 6.0a,b 1.0b 3.5a,b 3.0b
grape
Pomace 10 8.5a,b 6.25c 6.0e 5.75a 6.0c 6.0b 6.0b 0.0a 7.75b,c 5.25c 5.5a,b 2.0b 4.0b 3.75b,c
flour
15 7.25a 6.25c 8.0c 6.0a,b 6.0c,d 5.75a,b 7.0f 1.0b 7.25b,c 5.25c 5.75a 3.0c 4.25b 4.5c
a
Attribute ranges (weak endpoint vs. intense endpoint).
Crust appearance: homogeneity of crust colour/ surface, inhomogeneous vs. homogeneous; crust colour, bright vs. dark brown.
Bread slice appearance: crust thickness, thin vs. thick; crumb pore homogeneity/ size, inhomogeneous vs. homogeneous/ small vs. big; crumb colour, white vs. brown.
Texture: cohesiveness, very cohesive vs. less cohesive; springiness, springless vs. springy; toughness, weak vs. intense; hardness, smooth vs. hard; crumbliness, weak vs. intense;
adhesivity, not sticky vs. sticky; sand feeling, smooth vs. sandy.
Data are medians. Data with different alphabets in the same column are significantly different (p<0.05).
 Šporin et al.

(a) Crumb odour*

8
Off-taste* 7 Sour odour*
6
5
4
Aftertaste* Sweet odour*
3
2 Control
1 M6
0
M10
Yeast flavour Flavour intensity*
M15

Bitter taste* Sweet taste*

Salty taste Sour taste*

(b) Crumb odour*

8
Off-taste* 7 Sour odour *
6
5
4
Aftertaste* Sweet odour*
3
2 Control
1 Z6
0
Z10
Yeast flavour Flavour intensity*
Z15

Bitter taste* Sweet taste*

Salty taste Sour taste*

Figure 3. Sensory evaluation of the bread prepared from wheat flour dough (control) and doughs with additions of 6%,
10% and 15% ‘Merlot’ grape pomace flour (a: M6, M10, M15, respectively) and additions of 6%, 10% and 15% ‘Zelen’
grape pomace flour (b: Z6, Z10, Z15, respectively). *p < 0.05.

GPF dough bread. These changes were similar to those
reported by Hoye and Ross (2011). The instrumentally
measured colour changes can be considered as notice-
able when the total colour difference (E) values are
>2 (Savadkoohi et al., 2014). In both the ‘Zelen’ and
‘Merlot’ GPF breads, compared to the control bread,
the E values were indeed >2. Bread with 15%
‘Merlot’ GPF had the highest E value, meaning that
the colour of 15% ‘Merlot’ bread was the most different
from the control bread. The lowest E value was seen
for the 6% ‘Zelen’ bread.
Sensory profiling
The results of the sensory profiling and the bread slice
properties and texture are presented in Table 4, with the
odour and taste properties shown in Figure 3. GPF
addition resulted in significant differences in crust
appearance and in all of the sensory attributes, except
yeast flavour and salty taste of the crumb, as compared
to the control. The colour of the crumb correlated with
the colorimetric measurements, while the colour of the
crumb and the crust correlated with GPF addition
(Figure 2).

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Food Science and Technology International 0(0)

Some of these data are in conflict with the results of
the instrumental texture analysis, which indicated that
GPF addition resulted in firmer crumb texture. Crumb
hardness was positively correlated with ‘Merlot’ GPF
addition, but not with ‘Zelen’ GPF addition. These
contradicting data might be a consequence of specific
differences between the instrumental analyses and
human perception.
The GPF addition influenced the porosity of the
crumb, such that it became more inhomogeneous. All
of the samples enriched with GPF were significantly
different in terms of all of the textural properties, as
compared to the control. The texture of the crumb
was stickier for 10% and 15% GPF addition. Similar
data were reported by Wang et al. (2007), who tested
the effects of a green tea extract on the quality of bread.
The springiness of all of the samples in the present
study was significantly different from the control
bread, although this did not reach significance
between the samples with the different GPF additions.
The GPF addition had negative effects on cohesive-
ness of the crumb for all ‘Merlot’ GPF additions,
while ‘Zelen’ GPF addition only affected cohesiveness
at 15% addition. Increased GPF addition decreased
the toughness of the crust, with slightly more evident
effects at 10% and 15% ‘Zelen’ GPF addition. Similar
data were also reported in a study by Mildner-
Szkudlarz et al. (2011), where gumminess of breads
significantly increased with increased levels of their
grape by-product. Sand feeling increased with
increased GPF addition in the present study, whereby
‘Zelen’ GPF addition resulted in less sand feeling than
‘Merlot’ GPF addition. This might be due to the tech-
nology of the red wine production, where the ‘Merlot’
grape pomace had a higher proportion of seeds versus
skins, compared to the ‘Zelen’ grape pomace. Higher
proportion of seeds might also be the reason for the
evident sand feeling in the samples that contained
‘Merlot’ GPF.
The GPF addition had significant influence on fla-
vour and odour intensities, and on aftertaste and off-
taste of the bread. Aftertaste and off-taste were more
pronounced in ‘Merlot’ GPF breads. The acidity of the
bread was increased by GPF addition, with higher acid-
ity for ‘Merlot’ GPF addition than for ‘Zelen’ GPF
addition. At the same time, odour became more
intense, and in particular, sourer. Bitterness of the
bread correlated with GPF addition. Bitterness and
astringency might have been caused by the flavan-3-
ols in the GPF, which include (þ)-catechin, ()-epica-
techin, and especially proanthocyanidins (Hoye and
Ross, 2011; Kielhorn and Thorngate, 1999). Sweet
taste was indicated only for 6% ‘Zelen’ GPF addition
and for 15% ‘Merlot’ GPF addition. The GPF addition
had greater effects on sweet odour than on taste. All of
the samples except that with 10% ‘Merlot’ GPF add-
ition had significantly stronger sweet odour, compared
to the control bread. These data are similar to those
reported in earlier studies for grape seed flour (Hoye
and Ross, 2011) and a green tea extract (Wang et al.,
2007).
TPC and antioxidant activity
The TPC of the bread samples is given in Table 5, as
GAE equivalents (mg GAE/g sample, on a dry mass
basis). ‘Merlot’ GPF breads showed slightly higher
TPC than ‘Zelen’ GPF breads, although this difference
did not reach statistical significance. The TPCs of these
bread samples were positively correlated with GPF

Table 5. Total phenolic content and antioxidant activities of grape pomace flour and bread prepared from the wheat flour
dough (control) and breads with ‘Merlot’ or ‘Zelen’ grape pomace flour additions (6%, 10%, 15%).a

Antioxidant assay
Sample/ Total phenolic
addition content DPPH FRAP
Condition (%) (mg GAE/g dw) (mg GAE/g dw) (mg TE/100 g dw)

Grape pomace flour Merlot 32.73  0.84c 88.30  0.06d >2775

Zelen 28.29  3.20c 89.31  0.35d 2479.19  419.2
Control bread None <0.69 0.99  2.49a 45.32  5.45a
Bread with ‘Merlot’ grape 6 2.99  0.37a 16.65  3.09b 590.53  57.74b
pomace flour 10 4.11  0.12b 21.79  2.30b 829.10  13.38b,c
15 5.92  0.53b 31.30  6.15d,b 1204.95  80.50bc
Bread with ‘Zelen’ grape 6 2.42  0.41a 10.26  2.56b,c 454.76  67.39a,b
pomace flour 10 3.06  0.11a,b 21.06  4.39b 602.36  23.53b
15 3.65  0.75ab,b 20.32  7.59b 769.36  182.71b,c
a
Data are means  standard deviation (n ¼ 3). Data with different alphabets in the same column are significantly different (p < 0.05).

10

addition. The bread from the 15% ‘Merlot’ GPF add-
ition showed the highest TPC (5.92  0.53 mg GAE/g
dry mass), followed by 10% ‘Merlot’ GPF addition
(4.11  0.12 mg GAE/g dry mass), although the differ-
ence was not statistically significant. The 6% ‘Zelen’
GPF bread showed the lowest TPC (2.42  0.41 mg
GAE/g dry mass).
The total antioxidant activity determined by the
DPPH assay is also expressed as GAE equivalents
(mg GAE/g sample, on a dry mass basis; Table 5).
There were no differences in the DPPH total antioxi-
dant activities between the ‘Zelen’ and ‘Merlot’ GPFs.
The total antioxidant activity of the bread with 15%
‘Merlot’ GPF addition (31.30  6.15 mg GAE/g dry
mass) was the highest, although again, the differences
here between the various GPF additions were not stat-
istically significant. The total antioxidant activity of all
these supplemented breads was significantly higher than
that of the control bread.
The total antioxidant activity determined by the
FRAP assay for the flours and breads is expressed as
mg TE/100 g, on a dry mass basis (Table 5). These data
show that ‘Merlot’ GPF had the highest total antioxi-
dant activity, which was, however, out of the range for
this assay; ‘Zelen’ GPF showed total antioxidant activ-
ity of 2479.19  419.2 mg TE/100 g dry mass.
Upon addition to the dough, 15% ‘Merlot’ GPF
bread showed the highest antioxidant activity
(1204.95  80.5 mg TE/100 g dry mass), followed by
10% ‘Merlot’ GPF bread (829.1  13.38 mg TE/100 g
dry mass), although these were not significantly differ-
ent. The 6% ‘Zelen’ GPF bread showed the lowest total
antioxidant activity (454.76  67.39 mg TE/100 g dry
mass).
These antioxidant activities (i.e. for DPPH, FRAP)
were correlated with the TPC, a trend that was also
seen for grape seed flour addition to bread by Hoye
and Ross (2011). The correlation analysis showed posi-
tive and significant correlations between TPC and
FRAP of the breads (r ¼ 0.987; p < 0.01), TPC and
DPPH of the breads (r ¼ 0.941; p < 0.01), and FRAP
and DPPH of the breads (r ¼ 0.941; p < 0.01). These
antioxidant activities and the TPC of the GPF breads
were significantly higher than for the control bread,
which is in agreement with previous findings where
bread was fortified with ginger powder, grape seed
flour, turmeric, grape by-products and grape seed
extract (Hoye and Ross, 2011; Lim et al., 2011;
Mildner-Szkudlarz et al., 2011; Peng et al., 2010).
Of note, the phenolics are not very heat stable and
thus the reactive compounds and the baking process
might have resulted in heat damage to the phenolic
compounds (Lim et al., 2011; Peng et al., 2010).
Indeed, this might well be the reason that not all of
the effects seen here on the enriched bread increased
Šporin et al.
proportionally with the GPF additions. We have to
stress that in bakery products, the Maillard reaction
has an important role. Maillard reaction products
have been reported to have antioxidant activities
through scavenging oxygen radicals, copper and Fe2þ
chelators. Previous studies reported that the antioxi-
dant capacity of the final baked product might derive
from the intrinsic phenolic compounds of the flour, the
added phenolic ingredients, the other ingredients that
naturally contain phenolics, the intermediate phenolic
products that can be generated during baking (e.g. via
the Maillard reactions), the thermal-induced degrada-
tive products, and the polyphenol–polysaccharides
complexes (Gawlik-Dziki et al., 2013; Sivam et al.,
2010).
CONCLUSIONS
These rheological, textural and sensory analyses show
significant impact of GPF addition on the properties of
dough and the bread as the final product. The results of
the farinograph analysis show that the GPF cultivar had
a major impact on the rheological properties of the
dough. The ‘Merlot’ GPF addition increased develop-
ment time, stability and farinograph quality number,
which might explain the higher volume of the ‘Merlot’
GPF bread. The GPF concentration was negatively cor-
related with bread brightness and firmness. The ‘Merlot’
GPF addition resulted in decreased dough water
absorption. The ‘Merlot’ GPF addition had greater
negative impact on the bread firmness than the ‘Zelen’
GPF. More intensive sour taste and sand feeling in the
mouth, as well as stronger after-taste and off-taste, was
provoked by the ‘Merlot’ GPF additions. Both GPF
cultivars showed positive correlation with phenolic con-
tent and antioxidant activity in the final product; how-
ever, the phenolic contents and antioxidant activities
were higher when ‘Merlot’ GPF was added.
These data allow us to conclude that GPF is an
excellent nutritional enrichment source for bread.
Based on the sensory evaluation, the ‘Zelen’ cultivar
would be preferable. As consumer acceptance was not
a part of the present study, we cannot define the con-
sumer responses to these enriched breads, and thus this
aspect remains open for future studies. On the other
hand, the demand for products with clear definitions
of their contents, known as ‘clean label products’, is
increasing. From this point of view, the positive effects
of the ‘Merlot’ GPF on the dough rheological proper-
ties are most promising.
DECLARATION OF CONFLICTING INTERESTS
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
11
Food Science and Technology International 0(0)
FUNDING
The author(s) disclosed receipt of the following financial sup-
port for the research, authorship, and/or publication of this
article: Ministry of Education, Science and Sport of Slovenia
grant (grant number: P4-0116) and the Ministry of Economic
Development and Technology of Slovenia grant (grant
number: 3211-11-000465).
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