Journal of Cereal Science 79 (2018) 518e526

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

Influence of different levels of sodium chloride and of a reduced-

sodium salt substitute on volatiles formation and sensory quality of
wheat bread
Antonio Raffo*, Marina Carcea, Elisabetta Moneta, Valentina Narducci, Stefano Nicoli,
Marina Peparaio, Fiorella Sinesio, Valeria Turfani
CREA e Research Centre for Food and Nutrition, Via Ardeatina, 546-00178 Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The influence of sodium chloride or of the reduced-sodium salt substitute Pansalt® (NaCl 57%, KCl 28%,
Received 13 September 2017 MgSO4 12%, lysine hydrochloride 2%, silica 1%, iodine 0.0036%) at two addition levels, 1.5% and 3% (on
Received in revised form flour weight), on volatiles formation and sensory profile of yeasted wheat bread was investigated.
22 December 2017
Volatiles were determined by HS-SPME/GC-MS. An increased level of NaCl was associated to a reduced
Accepted 23 December 2017
Available online 27 December 2017
formation of 2-phenylethanol, a key odorant contributing to a yeast-like note. In contrast, at the 3% NaCl
level the formation of most of Maillard reaction volatile products was promoted. Accordingly, sensory
analysis highlighted a less intense yeasty note in the crumb, as well as more intense toasted aroma and
Chemical compounds studied in this article:
2-Phenylethanol (PubChem CID: 6054)
flavour in the crust of bread with 3% NaCl with respect to the 1.5% counterpart. Bread with 3% NaCl was
3-Methyl-1-Butanol (PubChem CID: 31260) also characterized by more bitter crust and more even crumb structure. Increasing the addition level of
2-MethylButanal (PubChem CID: 7284) Pansalt® from 1.5 to 3% produced similar effects on bread sensory attributes than NaCl. However, the
3-MethylButanal (PubChem CID: 11552) substitution by Pansalt® was not able to maintain the same level of perceived saltiness as NaCl at both
Methional (PubChem CID: 18635) addition levels. In particular, Pansalt® at 3% produced a perceptible increase in bitter taste and aftertaste
(E)-2-Nonenal (PubChem CID: 5283335) in the crust.
2,3-Butanedione (PubChem CID: 650) © 2018 Elsevier Ltd. All rights reserved.
2-Furfural (PubChem CID: 7362)
3-Ethyl-2,5-Dimethylpyrazine (PubChem
CID: 25916)
2-Ethyl-3,5-Dimethylpyrazine (PubChem
CID: 26334)

Keywords:
Wheat bread
Salt
Volatiles
Sensory quality

1. Introduction
It is widely agreed that the reduction of dietary sodium intake
can have a significant role in the control of blood pressure, which in
turn is considered one the major modifiable factors in the devel-
opment of cardiovascular and related diseases (Susic and Frohlich,
2012). Thus a number of actions have been recently undertaken to
reduce the amount of sodium chloride in foods (Strazzullo et al.,
2012). Bread, along with other bakery products, represents one of
* Corresponding author.
E-mail address: antonio.raffo@crea.gov.it (A. Raffo).
the major sources of sodium chloride in the daily Western diet, so
many initiatives aimed at lowering salt intake have focused on the
reduction of salt content in bread (Quilez and Salas-Salvado, 2012).
Sodium chloride plays an obvious role in determining the sen-
sory properties of bread but it also has important technological
functions in the breadmaking process, by influencing the devel-
opment of the gluten structure, the fermentation process and the
level of water activity in the baked product (Cauvain, 2007). Thus to
pursue the goal of reducing sodium levels in bread, it is of para-
mount importance to achieve a thorough understanding of all
functions of salt, including both techno-functional and sensory
aspects (Silow et al., 2016).
Several strategies have been proposed to reduce the salt content

https://doi.org/10.1016/j.jcs.2017.12.013
0733-5210/© 2018 Elsevier Ltd. All rights reserved.
 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526
of bread. A stepwise reduction of sodium chloride in time has been
applied to slowly reduce salt content in recipes without the con-
sumers' notice (Quilez and Salas-Salvado, 2012; Silow et al., 2016).
Salt content in bread marketed in Italy ranges from 0.7 to 2.3%
(corresponding to 1.2e3.1% on a dry matter basis, d.m.) for artisanal
bread and from 1.1 to 2.2% (1.6e3.2% d.m.) for industrial bread ac-
cording to a national survey carried out in 2009e2011 (Carcea et al.,
2016). Lynch et al. (2009) report that a salt reduction up to 0.3e0.6%
would be feasible without a significant deterioration in rheological
properties or performance during bread manufacturing. However,
the effect of salt reduction on the organoleptic properties is still a
critical factor for consumer acceptance (Lynch et al., 2009; Pflaum
et al., 2013). Use of salt replacers, particularly potassium salts, or
taste enhancers has been proposed to improve flavour quality of
foods with reduced salt. However, potassium salts alone have been
reported to give bitter and metallic off-tastes (Sinopoli and Lawless,
2012). For this reason, salt mixtures including sodium, potassium
and magnesium together with aminoacids such as lysine have been
experimented and patented to mask the mentioned unpleasant
taste originating from potassium chloride (see Pansalt®). Specific
data on the use of commercial salt mixtures or taste enhancers on
bread are scarce (Silow et al., 2016).
This study was part of the research project EUSAL aimed at
investigating strategies for salt reduction in breadmaking. The in-
fluence of different levels of sodium chloride or of the above
mentioned commercial salt substitute Pansalt® on volatiles for-
mation and sensory profile of wheat bread was investigated and the
results are presented and discussed in this paper. The selected
levels of addition (1.5 and 3% on flour weight) corresponded
approximately to the most common and the maximum level
observed in the above mentioned national survey. Therefore the
1.5% level was selected as the reference one.
2. Materials and methods
2.1. Preparation of bread samples
Loaves of bread were prepared from a commercial wheat flour
(Horeca brand, type 0 according to the Italian flour classification).
This flour was found to have 12.8% moisture, 0.63% d.m. ash, 10.5%
d.m. total protein, 0.8% d.m. fat, 3.2% d.m. total dietary fibre, 188
alveographic W (analyses were performed according to the ICC
methods n. 104/1, 105/2, 107/1, 110/1, 121, 136, 156 (ICC, 2003)). All
ingredients, apart from Pansalt®, were commercial products bought
on the market. Pansalt® is a patented reduced-sodium salt substi-
tute composed of NaCl (57%), KCl (28%), MgSO4 (12%), lysine hy-
drochloride (2%), silica (1% added as anticaking agent) and iodine
(0.0036%) and was kindly provided by Oriola-Oy (Espoo, Finland).
The baking method was an adaptation of the ICC Method n. 131
“Method for Test Baking of Wheat Flours” (ICC, 2003), in that so-
lution 1 was not used, i.e. malt and ascorbic acid were not added to
keep the recipe simple. Practically, a dough was made by mixing
1 kg flour (14% moisture basis, m.b.), with optimum water quantity
as determined by means of the Farinograph (according to ICC
Method n. 115/1 (ICC, 2003)), 18 g of compressed baker's yeast,
sucrose only in the amount prescribed by the ICC Method n. 131 to
activate yeast (3,6 g dissolved in part of the water so to obtain a 5%
sucrose solution), NaCl or Pansalt® at 1.5 or 3% on flour weight.
Mixing and proofing times, scaling and baking conditions were as
described by the ICC Method n. 131. A planetary mixer was used
(mod. Quick 20 by Sottoriva, Marano, Italy) and the loaves were
baked in a professional oven (model FX61G3, Angelo Po, Carpi MO,
Italy) at 220
C for about 30 min. Four loaves were prepared for each
batch: 2 were taken for volatile analysis and 2 for sensory
evaluation.
519
2.2. Analysis of volatile compounds
The approach developed in our laboratory and previously
described (Raffo et al., 2015) was adopted. Thus the following
samples were collected from the two replicate bread loaves: (i)
crust only, to achieve the highest sensitivity in the analysis of this
part of bread loaf, and (ii) whole slices, mainly formed by crumb,
which allowed to sample in a repeatable way both the crumb and
crust portion of the loaf. To collect these samples, bread loaves were
cooled at room temperature for 1 h and then cut in slices. Crust
samples were obtained by cutting crosswise a bread slice and col-
lecting 1 cm of its outer part. Then, about 60 g of whole slice sample
and 30 g of crust sample were frozen with liquid nitrogen and
ground by a laboratory grinding device (Ika, Staufen, Germany) to
give a powder that was stored at 70
C until analyses.
Volatiles were determined on both crust and whole slice sam-
ples by a semi-quantitative HS-SPME/GC-MS method. A fixed
amount (0.25 g) of bread powder, from whole slice or crust samples,
was placed in a 15 mL vial for SPME and 5 mL of the extraction
solution were added. The extraction solution was daily prepared by
placing 100 mL of the mixed internal standards aqueous solution in
a glass flask and then adding a NaCl 20% aqueous solution (pH
adjusted to 3 by addition of a 0.05 M aqueous solution of citric acid)
to a final volume of 50 mL. The mixed internal standards aqueous
solution was prepared by mixing the following volumes of the stock
methanol solutions of each internal standard in a 100 mL volu-
metric flask (Table 1S), 100 mL of 2-ethyl-2-butenal (4.35 mg mL1),
100 mL of 4-methyl-2-pentanol (4.04 mg mL1), 100 mL of 3-octen-
2-one (0.69 mg mL1), 500 mL of 1-(2-furyl)-acetone
(2.21 mg mL1), 1 mL of cis-7-decen-1-al (3.37 mg mL1), 50 mL of
5-isobutyl-2,3-dimethyl pyrazine (3.70 mg mL1), 100 mL p-tol-
ualdehyde (3.06 mg mL1), 100 mL of 1-phenyl-2-propanol
(4.86 mg mL1) and 1 mL of 3-acetyl pyridine (5.51 mg mL1), and
then adding deionized water to a total volume of 100 mL. The vial
containing the bread powder, the extraction solution and a mag-
netic stir bar was then capped with a PTFE/silicone septa for HS-
SPME and immersed in a water bath kept at 50
C. Then HS-SPME
extraction was carried out by exposing a 50/30 mm DVB/CAR/
PDMS fibre (Supelco, Sigma-Aldrich Italy) to the headspace of the
bread powder suspension for 60 min, while stirring at 700 rpm. At
the end of the extraction time the fibre was immediately inserted
into the gas chromatograph split-splitless injection port, for the
desorption step, and the GC run was started. GC/MS analyses were
performed on an Agilent 6890 GC 5973N MS system equipped with
a quadrupole mass filter for mass spectrometric detection (Agilent
Technologies, Palo Alto, CA). Desorption of extracted volatiles from
the fibre was carried out within the GC injector, operating by the
splitless mode, at 260
C for 5 min. GC separation was achieved on a
DB-Wax column (0.25 mm i.d.  60 m, 0.5 mm film thickness; J&W,
Agilent Technologies, Palo Alto, CA) by setting the following chro-
matographic conditions: inlet temperature was 260
C; oven tem-
perature programme from 40
C (10 min) to 210
C at 4
C min1,
and then to 220
C (5 min) at 30
C min1 (total run time of
57.8 min); constant flow of He carrier gas was 2 mL min1 corre-
sponding to a linear velocity of 36 cm s1. The MS detector operated
in the electron ionisation mode at 70 eV; transfer line, source, and
quadrupole temperatures were set, respectively, at 220, 230, and
150
C. Detection was performed in the full scan mode, over the
mass range 30e200 amu, for identification purposes, and in the
single ion monitoring (SIM) mode for quantification purposes.
Identification of bread samples volatiles was accomplished by
comparison of linear retention indices (LRI) and mass spectra of
chromatographic peaks with those obtained by applying the above
described HS-SPME/GC-MS method to an aqueous solution of pure
reference compounds or with those reported in the literature and
520 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526
in the NIST/EPA/NIH Mass Spectra Library 2005. For the semi-
quantitative determination of each compound chromatographic
signals obtained by the SIM mode were used, calculating the ratio
of the peak area of the target analyte to the peak area of the most
appropriate internal standard (Table 1S). For each target analyte the
most appropriate internal standard was that one which gave rise to
the most repeatable ratio of peak areas. The level of target analytes
in bread samples was calculated based on calibration curves of
target pure compounds, built by analysing matrix-matched test
samples containing target pure compounds (Raffo et al., 2015).
Matrix-matched test samples were prepared by placing in the
extraction vial the same fixed amount of a deodorised bread
powder sample, instead of the bread powder sample itself, and by
spiking the pure compounds into the extraction solution at six
concentration levels. For some target analytes the calibration curve
of the most structurally similar target compound was used
(Table 2). A triplicate analysis was performed on the bread powder
obtained from each loaf, for each batch.
2.3. Sensory analysis
Bread samples were evaluated and compared by quantitative
descriptive analysis (Raffo et al., 2003). A panel of professional as-
sessors with several years of experience in quantitative descriptive
sensory evaluation of foods and bread, in particular, analyzed the
bread samples. Individual bread samples, prepared as whole slices
1 cm thick, were served to the panelists in plates coded with three-
digit random numbers at room temperature (20 ± 2
C) in an
environmentally controlled sensory laboratory, provided with in-
dividual sensory booths. The bread samples were served in
monadic order balanced across the panelists. The panelists agreed a
consensus list of 16 attributes for profiling and definitions for each
attribute (Table 2S). Intensity ratings were scored on a continuous
15 cm unstructured scale anchored at the low end (“not percep-
tible”, 0-value) and at the high end (“strong”, 9-value) for some
descriptors, whereas distinctive anchor terms were used for others
(Table 2S). The experiment was designed so that three analytical
replicates were given to each panelist for each breadmaking
replicate.
2.4. Statistical analyses
A Principal Component Analysis (PCA) was performed on the
dataset of volatile compounds determined on both bread crust and
whole slice samples. Datasets used for PCA were obtained by first
averaging volatile levels obtained by analytical replicate de-
terminations and then applying auto-scaling as pre-treatment. To
identify which volatiles were significantly affected by changes in
bread formulation univariate methods of data analysis were per-
formed. While the assumption of normality of data on volatiles was
confirmed, by the Shapiro-Wilk test, the assumption of homosce-
dasticity, according to the Levene test, was not. Therefore, statistical
significance of differences in volatile levels between bread formu-
lations was evaluated by the non-parametric Kruskal-Wallis test,
whereas the Mann-Whitney test was applied to pairs of data as a
post-hoc test of significance between pairs of results (Granato et al.,
2014).
Data from sensory analyses were checked for panel uniformity
and normality. Significance of differences for data having a normal
distribution was assessed by ANOVA, and mean values for each
bread formulation were compared by a multiple comparison
Duncan test to look for grouping. For data not having a normal
distribution significance of differences was assessed by the
Kruskal-Wallis test.
All statistical analyses, except for the Mann-Whitney test, were
performed by the XL Stat (ver. 2016.05; Addinsoft, New York, NY)
software package. The Mann-Whitney test was performed by the
PAST software (version 2.17).
3. Results and discussion
3.1. Effects of different levels of NaCl and Pansalt®on formation of
volatile compounds. PCA and univariate data analysis
Thirty five volatile compounds were determined, belonging to
most of the chemical classes of bread odorants (Birch et al., 2014;
Pico et al., 2015): 4 alcohols, 13 aldehydes, 2 ketones, 4 furans, 4
pyrroles, 6 pyrazines, 2 other heterocyclic compounds. The evalu-
ation of the level of volatiles determined in the crust and in the
whole slice (Tables 1 and 2) showed, as expected, higher levels of
Maillard reaction products, such as pyrazines, pyrroles and furans,
in the crust than in the whole slice. The higher levels in the crust of
the aldehydes 2-methylpropanal, 2- and 3-methylbutanal, phe-
nylacetaldehyde and the ketone 2,3-butanedione are consistent
with their formation during baking within the complex of the
Maillard reactions, as discussed below. In contrast, compounds that
are mainly formed during fermentation and accumulate within the
crumb (2-phenylethanol, 2-/3-methyl-1-butanol) were found at
higher levels in whole slice samples. A two-dimensional repre-
sentation of projections along the first two components resulting
from a PCA allowed for a first exploration of the impact of the
considered formulation changes on the whole volatile profile of
bread crust and whole slice samples (Figs. 1 and 2). In the case of
bread crust the first two PCs explained 73% of the total variance,
with PC1 and PC2 accounting for, respectively, 49% and 24%. Most of
volatile compounds are clearly grouped according to their route of
formation in bread. Compounds whose main route of formation is
linked to the Maillard reaction are grouped close to the positive axis
of PC1: pyrazines, pyrroles, the Strecker aldehydes 2-
methylpropanal, 2- and 3-methylbutanal, phenylacetaldehyde
(but not methional), the ketones 2,3-butanedione and 1-octen-3-
one, some furans (2-acetylfuran and 2 furfural) and other hetero-
cyclic compounds (2-acetylpyridine). The main products of the
Ehrlich pathway, 2-phenylethanol, 2-/3-methyl-1-butanol are
located on the opposite side, close to the negative axis of PC1.
Volatiles formed through the third most important route of aroma
formation in bread, i.e. lipid oxidation (most of aldehydes and 2-
pentyl furan) are grouped close to the positive axis of PC2. A
similar picture, with minor differences, is also given by the repre-
sentation of the PCA results obtained on the whole slice dataset,
where the first two PCs accounted for 74% of explained variance
(PC1 53% and PC2 22%). It is well known that some volatiles found
in bread can be formed by more than one route of formation, which
sometimes complicates the interpretation of effects of processing
factors: for example, the aldehydes 2-methylpropanal, 2- and 3-
methylbutanal, phenylacetaldehyde and methional can be formed
through the Ehrlich pathway during the fermentation step or can
result from the Strecker degradation during baking (Pico et al.,
2015). Similarly, 2,3-butanedione can be formed either through
the glycolysis of pyruvic acid during fermentation, or as a product of
the Maillard reaction occurring during baking (Pico et al., 2015),
whereas furans can be derived from both lipid oxidation and the
Maillard reaction (Parker, 2015a). Groupings of compounds high-
lighted by PCA representations suggest that in the conditions of the
present study the Strecker degradation, and not the Ehrlich
pathway, was likely the main route of formation of 2-
methylpropanal, phenylacetaldehyde, 2- and 3-methylbutanal,
because in the PCA graph they are clearly associated to the other
products of the Maillard reaction and located quite far away from
the Ehrlich pathway products. Similarly, and for the same reason,
 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526 521

Table 1
Levels of volatile compounds (mg kg1) as obtained by semi-quantitative determination on crust samples of breads with NaCl or Pansalt® at 1.5 or 3% addition level.a

Compound Bread salty ingredients p-valueb

NaCl 1.5% NaCl 3% PanSalt® 1.5% Pansalt® 3%

Alcohols
2-/3-methyl-1-butanol 2933 bc 2603 b 1980 a 1972 a <.001
2 -phenylethanol 2527 d 1995 c 1775 b 1676 a <.001
p-vinyl guaiacol 551 a 504 a 557 ab 585 b <.01
Aldehydes
2-methylpropanal d 151 a 311 c 220 b 255 bc <.001
2-methylbutanal 819 a 1048 b 964 ab 917 ab <.01
3-methylbutanal 512 a 693 b 601 ab 552 ab <.01
hexanal 892 ab 979 b 657 a 1113 b <.01
methional 23 a 21 a 29 b 24 ab <.01
benzaldehyde e 151 a 181 ab 154 a 178 b <.01
(E)-2-nonenal 234 b 227 ab 171 a 227 ab <.05
(E,Z)-2,6-nonadienal 7.6 ab 7.7 ab 6.0 a 8.6 b <.01
phenylacetaldehyde 114 a 192 b 144 ab 177 b <.001
(E,E)-2,4-nonadienal f 76 b 43 ab 54 a 84 b <.01
(E,Z)-2,4-decadienal f 18 b 14 a 12 a 20 b <.001
(E,E)-2,4-decadienal 119 b 81 ab 51 a 116 b <.001
tr-4,5-epoxy-(E)-2-decenal f 15 a 15 a 14 ab 19 b <.01
Ketones
2,3-butanedione 1223 a 1472 a 1320 a 1412 a <.05
1-octen-3-one 23 a 26 ab 26 ab 28 b <.05
Furans
2-pentyl furan g 270 c 133 b 91 a 372 d <.001
2-furfural 7836 a 16654 ab 11366 a 21376 b <.001
2-acetylfuran 536 a 980 c 778 b 1188 c <.001
Pyrroles
1-methylpyrrole 25 c 15 a 28 c 18 b <.001
2-acetyl-1-methylpyrrole 14 a 14 a 18 b 17 b <.001
1-furfuryl pyrrole 51 a 78 b 62 ab 80 b <.001
2-acetylpyrrole 730 a 914 ab 891 ab 1013 b <.001
2-formyl pyrrole h 376 a 630 c 486 b 578 c <.001
Pyrazines
2,5-dimethylpyrazine 126 a 237 bc 195 b 242 c <.001
2,6-dimethylpyrazine i 242 a 334 b 341 b 350 b <.01
2-ethylpyrazine i 439 a 585 b 598 b 640 b <.01
2,3-dimethylpyrazine i 151 a 203 b 206 b 227 b <.001
3-ethyl-2,5-dimethylpyrazine 14 a 34 c 21 b 35 c <.001
2-ethyl-3,5-dimethylpyrazine 14 a 30 c 22 b 33 c <.001
Other heterocyclic compounds
2-acetylpyridine 52 a 59 ab 60 b 59b <.01
g-nonalactone 463 c 175 a 147 a 370 b <.001

Notes.
a
Mean values obtained by triplicate determinations on two distinct breadmaking replicates (two distinct loaves).
b
Significance of differences as assessed at different p values by the Kruskal-Wallis test.
c
Different letters denote significant differences between pairs of values according to the Mann-Whitney test (at p values < 0.05).
d
Semi-quantitative determination based on calibration curve of 2-methylbutanal.
e
Semi-quantitative determination based on calibration curve of phenylacetaldehyde.
f
Semi-quantitative determination based on calibration curve of (E,E)-2,4-decadienal.
g
Semi-quantitative determination based on calibration curve of 2-acetylfuran.
h
Semi-quantitative determination based on calibration curve of 2-acetylpyrrole.
i
Semi-quantitative determination based on calibration curve of 2,5-dimethylpyrazine.

the Maillard reaction rather than lipid oxidation seemed to be the
main biological source of 2,3-butanedione. As regards furans, 2-
furfural, 2-acetylfuran and 1-furfurylpyrrole were associated with
the formation of other Maillard reaction products, whereas 2-
pentylfuran, being located close to alkanals, 2-alkenals and 2,4-
alkanodienals in the PCA graphs, was likely derived from oxida-
tion of fatty acids, as previously reported (Parker, 2015b).
PCA representation of data obtained on both crust and whole
slice consistently shows that PC1 discriminated bread samples
based on their content of Maillard reaction volatile products
(associated to positive PC1 values) and of compounds formed
through the Ehrlich pathway (associated to negative PC1 values).
Thus PC1 effectively describes the influence of changes in formu-
lation on these two routes of formation of volatiles. When
comparing breads with NaCl, it is clear that a higher level of added
salt promoted the progress of the Maillard reaction and inhibited
the Ehrlich pathway. In particular, results from univariate data
analysis shows that increasing NaCl level from 1.5% to 3% produced
a significant decrease in the level of 2-phenylethanol (23%) and a
slight (not significant) decrease of 2-/3-methyl-1-butanol (7%) in
the whole slice (Table 2). These compounds are among the main
components of the volatile fraction of bread crumb, are major
contributors to its yeasty note and are formed through the Ehrlich
pathway, which is a secondary fermentation reaction carried out by
yeasts (Pico et al., 2015). Therefore, the reduction of their level in
the NaCl 3% bread can be plausibly explained by the inhibition ef-
fect of NaCl on yeast activity, which has been repeatedly reported in
the literature (Cauvain, 2007; He et al., 1992; Lynch et al., 2009) and
also observed in the present study by measurements of gas pro-
duction during dough fermentation (data not reported). In previous
studies, reduced levels of gas production in the dough, and thus of
yeast activity (Belz et al., 2017; He et al., 1992; Lynch et al., 2009),
522 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526

Table 2
Levels of volatile compounds (mg kg1) as obtained by semi-quantitative determination on whole slice samples of breads with NaCl or Pansalt® at 1.5 or 3% addition level.a

Compound Bread salty ingredients p-valueb

NaCl 1.5% NaCl 3% Pansalt® 1.5% Pansalt® 3%

Alcohols
2-/3-methyl-1-butanol 5794 bc 5342 b 4140 a 4344 a <.001
2 -phenylethanol 3031 d 2319 c 2133 b 1919 a <.001
Aldehydes
2-methylpropanal d 49 a 95 b 73 b 70 b <.001
2-methylbutanal 211 a 283 b 282 ab 217 ab <.05
3-methylbutanal 150 a 208 b 194 ab 157 ab <.05
hexanal 680 632 564 590 n.s.j
methional 12.1 12.2 11.5 12.0 n.s.
benzaldehyde e 132 139 136 151 n.s.
(E)-2-nonenal 64 a 79 bc 70 ab 85 c <.01
(E,Z)-2,6-nonadienal 2.3 a 3.0 a 2.6 a 3.2 a <.05
phenylacetaldehyde 63 a 118 c 83 b 93 c <.001
(E,E)-2,4-nonadienal f 32 a 34 ab 30 a 39 b <.01
(E,Z)-2,4-decadienal f 9a 10 ab 8a 11 b <.01
(E,E)-2,4-decadienal 78 a 71 a 57 a 106 b <.01
tr-4,5-epoxy-(E)-2-decenal f 6.3 8.9 7.8 7.8 n.s.
Ketones
2,3-butanedione 467 a 711 c 557 b 683 bc <.001
1-octen-3-one 10 a 17 c 9a 13 b <.001
Furans
2-pentyl furan g 152 b 89 a 86 a 244 c <.001
2-furfural 889 a 3776 bc 2702 b 3922 c <.001
2-acetylfuran 114 a 259 bc 241 b 329 c <.001
Pyrroles
1-methylpyrrole 4.0 a 3.5 a 6.3 b 2.9 a <.01
2-acetyl-1-methylpyrrole 3.3 ab 3.7 a 4.4 b 3.7 ab <.05
1-furfuryl pyrrole 9a 20 c 15 b 16 bc <.001
2-acetylpyrrole 151 a 243 b 229 b 254 b <.01
2-formyl pyrrole h 70 a 125 c 72 ab 117 bc <.01
Pyrazines
2,5-dimethylpyrazine 23 a 51 b 43 b 51 b <.01
2,6-dimethylpyrazine i 64 a 99 ab 112 b 94 ab <.01
2-ethylpyrazine i 54 a 91 b 92 b 94 b <.01
2,3-dimethylpyrazine i 27 a 48 b 48 b 49 b <.01
3-ethyl-2,5-dimethylpyrazine 2.8 a 8.1 c 5.1 b 7.3 c <.001
2-ethyl-3,5-dimethylpyrazine 2.7 a 6.7 c 5.0 b 6.9 c <.001
Other heterocyclic compounds
2-acetylpyridine 9a 14 b 13 b 12 ab <.01
g-nonalactone 255 c 110 a 102 a 203 b <.001

Notes.
a
Mean values obtained by triplicate determinations on two distinct breadmaking replicates (two distinct loaves).
b
Significance of differences as assessed at different p values by the Kruskal-Wallis test.
c
Different letters denote significant differences between pairs of values according to the Mann-Whitney test (at p values < 0.05).
d
Semi-quantitative determination based on calibration curve of 2-methylbutanal.
e
Semi-quantitative determination based on calibration curve of phenylacetaldehyde.
f
Semi-quantitative determination based on calibration curve of (E,E)-2,4-decadienal.
g
Semi-quantitative determination based on calibration curve of 2-acetylfuran.
h
Semi-quantitative determination based on calibration curve of 2-acetylpyrrole.
i
Semi-quantitative determination based on calibration curve of 2,5-dimethylpyrazine.
j
Not significant at p ¼ .05 by the Kruskal-Wallis test.

and less intense sensory perception of the yeasty note (Lynch et al.,
2009; Pflaum et al., 2013), were observed in bread with increased
NaCl levels. In the present study, the observation of a reduction in
the level of 2-phenylethanol, while confirming recently reported
similar results (Belz et al., 2017), provides a further insight into the
molecular basis of the reduced perception of the yeasty note.
Another effect of increasing NaCl level from 1.5% to 3% is the
enhanced formation in the crust of most of the Maillard products:
the key odorants pyrazines (3-ethyl-2,5-dimethylpyrazine, 2-ethyl-
3,5-dimethylpyrazine), the Strecker aldehydes (2-methylpropanal,
2- and 3-methylbutanal, phenylacetaldehyde), some furans (2-
furfural, 2-acetylfuran), and the pyrroles (1-furfurylpyrrole, 2-
acetylpyrrole, 2-fromylpyrrole) (Tables 1 and 2). Increased levels
of NaCl in the dough have been observed to promote crust colour
formation in bread and breakfast cereals (Moreau et al., 2009; Silow
et al., 2016) and therefore have been supposed to increase the rate
of the Maillard reaction. One possible explanation was that
increased levels of NaCl can promote the formation of melanoidins
by the Maillard reaction (Silow et al., 2016), an effect that resembles
that one observed in the present study on the volatile products of
the Maillard reaction. NaCl addition may increase the rate of the
Maillard reaction through several mechanisms: by influencing
water activity, moisture content and pH of the dough, or by a
plasticising effect on the food matrix during the backing step,
which could improve the mobility of the reactants of the Maillard
reaction, thus favouring the progress of the reaction (Moreau et al.,
2009). Alternatively, it has been suggested that the lowered yeast
activity observed during dough fermentation and associated to the
addition of NaCl, could leave an increased amount of sugars not
metabolised at the end of fermentation and thus an increased pool
of them could be available for the formation of browning Maillard
products (Skobranek, 1998). In the present study, all of these
 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526 523

Fig. 1. PCA of volatiles determined on bread crust samples. Bi-plot (scores and loadings plot) of the first two PCs.
Note: Data points denoted by NaCl 1,5 and NaCl 3 corresponded to bread samples with NaCl at 1.5% and 3%, respectively. Data points denoted by PS 1,5 and PS 3 corresponded to
bread samples with Pansalt® at 1.5% and 3%, respectively. The last figure in the label denotes each of the two breadmaking replicates (two distinct loaves).

mechanisms could have contributed to the observed increase of
volatile Maillard products. Only a direct measurement of an effect
on sugar levels in the dough at the end of the fermentation step
would support the hypothesis that the enhanced level of Maillard
volatiles could have been due to a NaCl effect on the yeasts activity
and thus on the concentration of precursors of the Maillard
reaction.
PCA results show that a similar influence on the Ehrlich pathway
and the Maillard reaction products was also observed when
comparing the two breads with Pansalt®, even though the effect of
the increased addition level was less marked (Figs. 1 and 2). A
significant reduction of the level of 2-phenylethanol, but not of 2-/
3-methyl-1-butanol, and an increase of many volatile Maillard re-
action products in the crust was also observed, even though the
impact was generally less marked than in the case of NaCl (Tables 1
and 2).
As regards the effect of substitution of NaCl with Pansalt®, the
PCA representation suggests that at 1.5% it tended to favour the
formation of Maillard reaction products and to inhibit the Ehrlich
pathway, whereas at the level of 3% this effect was less pronounced
(Figs. 1 and 2). Univariate analysis shows that at both levels of
addition, substitution of NaCl by Pansalt® is accompanied by a
significant reduction of 2-phenylethanol and 2-/3-methyl-1-
butanol levels (Tables 1 and 2), but it is not possible to state here
whether this effect was mediated by an influence of the different
inorganic salts composition on yeasts activity. In contrast, the
substitution of NaCl by Pansalt®, promoted the formation of only
few of the Maillard products (3-ethyl-2,5-dimethylpyrazine, 2-
ethyl-3,5-dimethylpyrazine, 2-acetylfuran, 2-formylpyrrole), and
only at the 1.5% addition level. The presence of lysine in the
formulation of Pansalt®, corresponding to a concentration of 1.6
and 3.3 mmol of lysine per Kilogram of flour, for addition of Pan-
salt® at 1.5 and 3%, respectively, could in principle favour the for-
mation of Maillard products with respect to the formulations with
NaCl. However, the presence of an effect only at the lower addition
level but not at 3%, suggested that the presence of lysine in the salt
replacer was not likely the factor that could explain the increased
formation of Maillard compounds in bread with Pansalt® at 1.5%.
Considering the second Principal Component in the PCA graph,
it can be seen that it discriminates bread samples based on their
level of lipid oxidation products (associated to positive PC2 values),
highlighting a lower level of this group of compounds in the bread
with 1.5% Pansalt® with respect to all the other breads. This effect
seems not to be easily explainable based on experimental data
collected in the present study.
PCA representations also allowed for a visual evaluation of the
extent of variation of the volatile profile associated to the experi-
mental procedure for breadmaking, as measured by the distance in
the graph between the two replicates for each bread formulation.
3.2. Effects of different levels of NaCl and Pansalt® on bread sensory
profile
Besides the obvious more intense perception of saltiness,
significant changes in other sensory attributes were produced by
the increase of NaCl from 1.5 to 3% (Table 3). A higher level of salt
524 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526

Fig. 2. PCA of volatiles determined on bread whole slice samples. Bi-plot (scores and loadings plot) of the first two PCs.
Note: Data points denoted by NaCl 1,5 and NaCl 3 corresponded to bread samples with NaCl at 1.5% and 3%, respectively. Data points denoted by PS 1,5 and PS 3 corresponded to
bread samples with Pansalt® at 1.5% and 3%, respectively. The last figure in the label denotes each of the two breadmaking replicates (two distinct loaves).

Table 3
Sensory profile of breads with NaCl or Pansalt® at 1.5 or 3% addition level.

Sensory descriptor Bread salty ingredientsa p-valueb

® ®
NaCl 1.5% NaCl 3% Pansalt 1.5% Pansalt 3%

Aroma, taste and flavour

Bakery aroma 6.46 6.27 6.31 6.03 n.s.c
Yeast aroma (crumb) 2.77 a 1.47 b 3.09 a 1.13 b <0.001
Toasted aroma (crust) 6.49 b 7.35 a 6.28 b 6.79 ab <0.01
Sweet (crumb) 4.83 b 2.18 d 5.69 a 3.56 c <0.001
Salty (crumb) 2.38 c 6.66 a 1.15 d 5.69 b <0.001
Bitter (crust) 2.62 c 5.38 b 2.62 c 6.39 a <0.001
Global flavour 6.94 7.12 6.78 7.14 n.s.
Toasted flavour 5.39 b 6.55 a 5.19 b 6.66 a <0.001
Bitter aftertaste (crust) 0.39 c 2.33 b 0.16 c 3.25 a <0.001
Texture and mouthfeel
Crumb moisture 7.02 7.43 7.16 6.86 n.s.
Hardness (crust) 6.65 6.98 6.47 6.78 n.s.
Crispness (crust) 6.37 b 7.03 a 6.39 b 7.04 a <0.01
Cohesiveness (crumb) 6.33 6.88 6.30 6.88 n.s.
Elasticity (crumb) 6.12 b 6.51 ab 6.23 ab 6.76 a <0.05
Crumb holes structure 5.20 a 4.13 b 4.40 b 3.89 b <0.001
Crumb lightness 5.73 5.26 5.50 5.52 <0.05
a
Mean values obtained on analytical replicates (6 assessors x 3 replicates) and two distinct breadmaking replicates, i.e. two distinct loaves (n ¼ 36).
b
For each descriptors, significance of differences for data having a normal distribution was assessed by ANOVA, and different letters in a row denote significant difference
between bread formulations according to the multiple comparison Duncan test (at p < .05). For data not having a normal distribution significance of differences was assessed
by the Kruskal-Wallis test, and different letters denote significant differences between pairs of values according to the Mann-Whitney test (at p < .05).
c
Not significant at p ¼ .05 by the ANOVA or Kruskal-Wallis test.
 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526
was associated with a less intense yeasty note, as perceived on
the crumb. A similar effect was previously observed on wheat
bread by increasing salt level from 0 to 2%, based on flour
(Pflaum et al., 2013), and it is consistent with effects of NaCl
increase on formation of 2-phenylethanol and 2-/3-methyl-1-
butanol found in the present study (Section 3.1). A higher level
of NaCl was also accompanied by more intense toasted flavour
and aroma, as perceived on the crust. Also this effect was
consistent with results of volatile profiling, the more intense
toasted notes corresponding to the higher concentration of the
Maillard volatile products observed in the 3% NaCl bread
(Table 1). The increase of toasted notes perceived on bread crust
paralleled an even more marked increase of two other taste
sensations, also perceived on the crust, such as bitter taste and
aftertaste. An opposite effect of NaCl on bitterness was previously
observed, but in that case the sensory evaluation was performed
on crumb instead of crust (Pflaum et al., 2013). In another study
no effect was observed on bitterness, but it was not specified
whether crust or crumb samples were analyzed (Lynch et al.,
2009). Whether the increase in the bitter sensations observed
in the present study could be due to higher levels of non volatile
products of the Maillard reaction could be the object of further
investigations. The crumb of bread with 3% NaCl was also
perceived as less sweet than the 1.5% counterpart. In contrast,
salt increase has been sometimes reported in the past to have a
promoting effect on bread sweetness (Miller and Hoseney, 2008),
whereas in recent experimental studies no effect of NaCl increase
was observed on sweetness (Lynch et al., 2009; Pflaum et al.,
2013). In the case of Pflaum et al. (2013), the addition of a
higher amount of sucrose, 1% based on the flour weight, than in
the formulation used in the present study (0.36%) could have
levelled off the perception of sweetness. As regards the effects on
bread texture, the results reported in the present study
confirmed that an increase in NaCl level gave rise to a more even
crumb structure, with smaller and more homogeneous pores.
This effect had been previously observed and explained on the
basis of the influence of NaCl addition on gluten physical prop-
erties and the resulting enhanced stability of water vapor bub-
bles during baking (Lynch et al., 2009; Silow et al., 2016). In
addition, in the present study a slight (not significant) increase
was also observed at the 3% level on crumb elasticity and crust
crispness.
Increasing the addition level of Pansalt® from 1.5 to 3% had a
higher impact on some bread sensory attributes than that pro-
duced by the increase of NaCl (Table 3). Interestingly, substitu-
tion by Pansalt® was not effective in maintaining the level of
saltiness (as perceived in the crumb) of the NaCl counterpart at
both addition levels. It means that the inorganic salts mixture
and other taste enhancers contained in PanSalt® did not fully
offset the reduction in saltiness due to the lower amount of NaCl
in the Pansalt® formulation. Moreover, the Pansalt® breads were
significantly sweeter than the NaCl counterparts and, considering
all bread samples, sweetness resulted to be inversely related to
saltiness (Pearson's correlation coefficient ¼  0.87; p < .001).
The crust of the 3% Pansalt® bread was perceived as more bitter
than the NaCl counterpart. This last effect might be due to the
presence in the Pansalt® formulation of KCl and magnesium sul-
phate, which are both known to be responsible for a metallic-bitter
aftertaste that becomes more perceptible at increasing levels of
addition (Silow et al., 2016). This suggested that the inclusion of
lysine and iodine in the formulation was not effective in masking
the bitter note produced by these two salts, at least at the highest
level of addition of the salt substitute. As a general remark the level
of addition, rather than the type, of the salty ingredient seemed to
be the major source of variation in the sensory profile.
525
4. Conclusions
In this study for the first time the influence of NaCl addition on
the formation of bread aroma compounds was investigated in
detail, highlighting a significant impact of the addition level on
some of the most important routes of formation of volatiles.
Increasing NaCl level from 1.5 to 3% gave rise to a significant
reduction of the yeasty smelling crumb odorant 2-phenylethanol,
while promoting the formation of many volatile products of the
Maillard reaction. In addition, significant changes in several sen-
sory attributes besides saltiness, such as aroma, other taste and
texture attributes were observed. Some of these additional effects
can be related to the influence of NaCl level on yeast activity during
fermentation, on gluten physical properties and reactions occurring
during baking. Moreover, data were provided on the effects of the
use of a commercial salt substitute on volatiles formation and
sensory quality. Substitution of NaCl by the mixture of inorganic
salts and taste enhancers present in Pansalt® was only partially
effective in maintaining the saltiness elicited by NaCl, while giving
rise to perceptible increase in bitter taste and aftertaste in crust at
the 3% addition level.
Funding
This work was supported by the Italian Ministry of Agriculture
and Forestry [EUSAL Project].
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgment
Thanks are due to Mr. L. Bartoli for technical help in bread
making.
Appendix A. Supplementary data
Supplementary data (Tables 1S and 2S) associated with this
article can be found in the online version, at https://doi.org/10.
1016/j.jcs.2017.12.013.
References
Belz, M., Axel, C., Beauchamp, J., Zannini, E., Arendt, E., Czerny, M., 2017. Sodium
chloride and its influence on the aroma profile of yeasted bread. Foods 6, 66.
https://doi.org/10.3390/foods6080066.
Birch, A.N., Petersen, M.A., Hansen, Å.S., 2014. Aroma of wheat bread crumb. Cereal
Chem. 91, 105e114.
Carcea, M., Narducci, V., Turfani, V., Sinesio, F., Raffo, A., Bianchi, P.G., Losi, M.,
Zecchinelli, R., Delogu, C., Andreani, L., 2016. Pane buono con poco sale. Molini
d’Italia 12, 36e42.
Cauvain, S.P., 2007. Reduced salt in bread and other baked products. In: Kilcast, D.,
Angus, F. (Eds.), Reducing Salt in Foods. Practical Strategies. Woodhead Pub-
lishing Limited, Cambridge, pp. 283e295.
Granato, D., de Araújo Calado, V.M., Jarvis, B., 2014. Observations on the use of
statistical methods in food science and technology. Food Res. Int. 55, 137e149.
https://doi.org/10.1016/j.foodres.2013.10.024.
He, H., Roach, R.R., Hoseney, R.C., 1992. Effect of nonchaotropic salts on flour bread-
making properties. Cereal Chem. 69, 366e371.
ICC (International Association for Cereal Science and Technology), 2003. Standard
Methods of the International Association for Cereal Science and Technology.
The Association, Vienna, Austria.
Lynch, E.J., Dal Bello, F., Sheehan, E.M., Cashman, K.D., Arendt, E.K., 2009. Funda-
mental studies on the reduction of salt on dough and bread characteristics.
Food Res. Int. 42, 885e891. https://doi.org/10.1016/j.foodres.2009.03.014.
Miller, R.A., Hoseney, R.C., 2008. Role of salt in baking. Cereal Foods World 53, 4e6.
Moreau, L., Lagrange, J., Bindzus, W., Hill, S., 2009. Influence of sodium chloride on
colour, residual volatiles and acrylamide formation in model systems and
breakfast cereals. Int. J. Food Sci. Technol. 44, 2407e2416. https://doi.org/
10.1111/j.1365-2621.2009.01922.x.
526 A. Raffo et al. / Journal of Cereal Science 79 (2018) 518e526
Parker, J.K., 2015a. Thermal generation of aroma. In: Parker, J.K., Elmore, J.S.,
Methven, L. (Eds.), Flavour Development, Analysis and Perception in Food and
Beverages. Woodhead Publishing, Cambridge, pp. 151e185.
Parker, J.K., 2015b. Introduction to aroma compounds in foods. In: Parker, J.K.,
Elmore, J.S., Methven, L. (Eds.), Flavour Development, Analysis and Perception
in Food and Beverages. Woodhead Publishing, Cambridge, pp. 3e30.
Pflaum, T., Konitzer, K., Hofmann, T., Koehler, P., 2013. Analytical and sensory studies
on the release of sodium from wheat bread crumb. J. Agric. Food Chem. 61,
6485e6494. https://doi.org/10.1021/jf4012906.

mez, M., 2015. Wheat bread aroma compounds in crumb and
Pico, J., Bernal, J., Go
crust: a review. Food Res. Int. 75, 200e215. https://doi.org/10.1016/
j.foodres.2015.05.051.
Quilez, J., Salas-Salvado, J., 2012. Salt in bread in Europe: potential benefits of
reduction. Nutr. Rev. 70, 666e678. https://doi.org/10.1111/j.1753-
4887.2012.00540.x.
Raffo, A., Pasqualone, A., Sinesio, F., Paoletti, F., Quaglia, G., Simeone, R., 2003. In-
fluence of durum wheat cultivar on the sensory profile and staling rate of
Altamura bread. Eur. Food Res. Technol. Z Lebensm. Unters. Forsch. 218, 49e55.
https://doi.org/10.1007/s00217-003-0793-1.
Raffo, A., Carcea, M., Castagna, C., Magrì, A., 2015. Improvement of a headspace solid
phase microextraction-gas chromatography/mass spectrometry method for the
analysis of wheat bread volatile compounds. J. Chromatogr. A 1406, 266e278.
https://doi.org/10.1016/j.chroma.2015.06.009.
Silow, C., Axel, C., Zannini, E., Arendt, E.K., 2016. Current status of salt reduction in
bread and bakery products e a review. J. Cereal. Sci. 72, 135e145. https://
doi.org/10.1016/j.jcs.2016.10.010.
Sinopoli, D.A., Lawless, H.T., 2012. Taste properties of potassium chloride alone and
in mixtures with sodium chloride using a check-all-that-apply method. J. Food
Sci. 77, S319eS322. https://doi.org/10.1111/j.1750-3841.2012.02862.x.
Skobranek, H., 1998. B€ ackereitechnologie, third ed. Handwerk und Technik,
Hamburg.
Strazzullo, P., Cairella, G., Campanozzi, A., Carcea, M., Galeone, D., Galletti, F.,
Giampaoli, S., Iacoviello, L., Scalfi, L., 2012. Population based strategy for dietary
salt intake reduction: Italian initiatives in the European framework. Nutrition,
Metabol. Cardiovasc. Dis. 22 (3), 161e166. https://doi.org/10.1016/
j.numecd.2011.10.004.
Susic, D., Frohlich, E.D., 2012. Salt consumption and cardiovascular, renal, and hy-
pertensive diseases: clinical and mechanistic aspects. Curr. Opin. Lipidol. 23 (1),
11e16. https://doi.org/10.1097/MOL.0b013e32834d9c52.