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Journal of Cereal Science 79 (2018) 518e526

Contents lists available at ScienceDirect

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 the major sources of sodium chloride in the daily Western diet, so
many initiatives aimed at lowering salt intake have focused on the
It is widely agreed that the reduction of dietary sodium intake reduction of salt content in bread (Quilez and Salas-Salvado, 2012).
can have a significant role in the control of blood pressure, which in Sodium chloride plays an obvious role in determining the sen-
turn is considered one the major modifiable factors in the devel- sory properties of bread but it also has important technological
opment of cardiovascular and related diseases (Susic and Frohlich, functions in the breadmaking process, by influencing the devel-
2012). Thus a number of actions have been recently undertaken to opment of the gluten structure, the fermentation process and the
reduce the amount of sodium chloride in foods (Strazzullo et al., level of water activity in the baked product (Cauvain, 2007). Thus to
2012). Bread, along with other bakery products, represents one of 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
* Corresponding author. aspects (Silow et al., 2016).
E-mail address: antonio.raffo@crea.gov.it (A. Raffo). 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 519

of bread. A stepwise reduction of sodium chloride in time has been 2.2. Analysis of volatile compounds
applied to slowly reduce salt content in recipes without the con-
sumers' notice (Quilez and Salas-Salvado, 2012; Silow et al., 2016). The approach developed in our laboratory and previously
Salt content in bread marketed in Italy ranges from 0.7 to 2.3% described (Raffo et al., 2015) was adopted. Thus the following
(corresponding to 1.2e3.1% on a dry matter basis, d.m.) for artisanal samples were collected from the two replicate bread loaves: (i)
bread and from 1.1 to 2.2% (1.6e3.2% d.m.) for industrial bread ac- crust only, to achieve the highest sensitivity in the analysis of this
cording to a national survey carried out in 2009e2011 (Carcea et al., part of bread loaf, and (ii) whole slices, mainly formed by crumb,
2016). Lynch et al. (2009) report that a salt reduction up to 0.3e0.6% which allowed to sample in a repeatable way both the crumb and
would be feasible without a significant deterioration in rheological crust portion of the loaf. To collect these samples, bread loaves were
properties or performance during bread manufacturing. However, cooled at room temperature for 1 h and then cut in slices. Crust
the effect of salt reduction on the organoleptic properties is still a samples were obtained by cutting crosswise a bread slice and col-
critical factor for consumer acceptance (Lynch et al., 2009; Pflaum lecting 1 cm of its outer part. Then, about 60 g of whole slice sample
et al., 2013). Use of salt replacers, particularly potassium salts, or and 30 g of crust sample were frozen with liquid nitrogen and
taste enhancers has been proposed to improve flavour quality of ground by a laboratory grinding device (Ika, Staufen, Germany) to
foods with reduced salt. However, potassium salts alone have been give a powder that was stored at 70  C until analyses.
reported to give bitter and metallic off-tastes (Sinopoli and Lawless, Volatiles were determined on both crust and whole slice sam-
2012). For this reason, salt mixtures including sodium, potassium ples by a semi-quantitative HS-SPME/GC-MS method. A fixed
and magnesium together with aminoacids such as lysine have been amount (0.25 g) of bread powder, from whole slice or crust samples,
experimented and patented to mask the mentioned unpleasant was placed in a 15 mL vial for SPME and 5 mL of the extraction
taste originating from potassium chloride (see Pansalt®). Specific solution were added. The extraction solution was daily prepared by
data on the use of commercial salt mixtures or taste enhancers on placing 100 mL of the mixed internal standards aqueous solution in
bread are scarce (Silow et al., 2016). a glass flask and then adding a NaCl 20% aqueous solution (pH
This study was part of the research project EUSAL aimed at adjusted to 3 by addition of a 0.05 M aqueous solution of citric acid)
investigating strategies for salt reduction in breadmaking. The in- to a final volume of 50 mL. The mixed internal standards aqueous
fluence of different levels of sodium chloride or of the above solution was prepared by mixing the following volumes of the stock
mentioned commercial salt substitute Pansalt® on volatiles for- methanol solutions of each internal standard in a 100 mL volu-
mation and sensory profile of wheat bread was investigated and the metric flask (Table 1S), 100 mL of 2-ethyl-2-butenal (4.35 mg mL1),
results are presented and discussed in this paper. The selected 100 mL of 4-methyl-2-pentanol (4.04 mg mL1), 100 mL of 3-octen-
levels of addition (1.5 and 3% on flour weight) corresponded 2-one (0.69 mg mL1), 500 mL of 1-(2-furyl)-acetone
approximately to the most common and the maximum level (2.21 mg mL1), 1 mL of cis-7-decen-1-al (3.37 mg mL1), 50 mL of
observed in the above mentioned national survey. Therefore the 5-isobutyl-2,3-dimethyl pyrazine (3.70 mg mL1), 100 mL p-tol-
1.5% level was selected as the reference one. 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
2. Materials and methods then adding deionized water to a total volume of 100 mL. The vial
containing the bread powder, the extraction solution and a mag-
2.1. Preparation of bread samples 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
Loaves of bread were prepared from a commercial wheat flour extraction was carried out by exposing a 50/30 mm DVB/CAR/
(Horeca brand, type 0 according to the Italian flour classification). PDMS fibre (Supelco, Sigma-Aldrich Italy) to the headspace of the
This flour was found to have 12.8% moisture, 0.63% d.m. ash, 10.5% bread powder suspension for 60 min, while stirring at 700 rpm. At
d.m. total protein, 0.8% d.m. fat, 3.2% d.m. total dietary fibre, 188 the end of the extraction time the fibre was immediately inserted
alveographic W (analyses were performed according to the ICC into the gas chromatograph split-splitless injection port, for the
methods n. 104/1, 105/2, 107/1, 110/1, 121, 136, 156 (ICC, 2003)). All desorption step, and the GC run was started. GC/MS analyses were
ingredients, apart from Pansalt®, were commercial products bought performed on an Agilent 6890 GC 5973N MS system equipped with
on the market. Pansalt® is a patented reduced-sodium salt substi- a quadrupole mass filter for mass spectrometric detection (Agilent
tute composed of NaCl (57%), KCl (28%), MgSO4 (12%), lysine hy- Technologies, Palo Alto, CA). Desorption of extracted volatiles from
drochloride (2%), silica (1% added as anticaking agent) and iodine the fibre was carried out within the GC injector, operating by the
(0.0036%) and was kindly provided by Oriola-Oy (Espoo, Finland). splitless mode, at 260  C for 5 min. GC separation was achieved on a
The baking method was an adaptation of the ICC Method n. 131 DB-Wax column (0.25 mm i.d.  60 m, 0.5 mm film thickness; J&W,
“Method for Test Baking of Wheat Flours” (ICC, 2003), in that so- Agilent Technologies, Palo Alto, CA) by setting the following chro-
lution 1 was not used, i.e. malt and ascorbic acid were not added to matographic conditions: inlet temperature was 260  C; oven tem-
keep the recipe simple. Practically, a dough was made by mixing perature programme from 40  C (10 min) to 210  C at 4  C min1,
1 kg flour (14% moisture basis, m.b.), with optimum water quantity and then to 220  C (5 min) at 30  C min1 (total run time of
as determined by means of the Farinograph (according to ICC 57.8 min); constant flow of He carrier gas was 2 mL min1 corre-
Method n. 115/1 (ICC, 2003)), 18 g of compressed baker's yeast, sponding to a linear velocity of 36 cm s1. The MS detector operated
sucrose only in the amount prescribed by the ICC Method n. 131 to in the electron ionisation mode at 70 eV; transfer line, source, and
activate yeast (3,6 g dissolved in part of the water so to obtain a 5% quadrupole temperatures were set, respectively, at 220, 230, and
sucrose solution), NaCl or Pansalt® at 1.5 or 3% on flour weight. 150  C. Detection was performed in the full scan mode, over the
Mixing and proofing times, scaling and baking conditions were as mass range 30e200 amu, for identification purposes, and in the
described by the ICC Method n. 131. A planetary mixer was used single ion monitoring (SIM) mode for quantification purposes.
(mod. Quick 20 by Sottoriva, Marano, Italy) and the loaves were Identification of bread samples volatiles was accomplished by
baked in a professional oven (model FX61G3, Angelo Po, Carpi MO, comparison of linear retention indices (LRI) and mass spectra of
Italy) at 220  C for about 30 min. Four loaves were prepared for each chromatographic peaks with those obtained by applying the above
batch: 2 were taken for volatile analysis and 2 for sensory described HS-SPME/GC-MS method to an aqueous solution of pure
evaluation. 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- performed by the XL Stat (ver. 2016.05; Addinsoft, New York, NY)
quantitative determination of each compound chromatographic software package. The Mann-Whitney test was performed by the
signals obtained by the SIM mode were used, calculating the ratio PAST software (version 2.17).
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 3. Results and discussion
most appropriate internal standard was that one which gave rise to
the most repeatable ratio of peak areas. The level of target analytes 3.1. Effects of different levels of NaCl and Pansalt®on formation of
in bread samples was calculated based on calibration curves of volatile compounds. PCA and univariate data analysis
target pure compounds, built by analysing matrix-matched test
samples containing target pure compounds (Raffo et al., 2015). Thirty five volatile compounds were determined, belonging to
Matrix-matched test samples were prepared by placing in the most of the chemical classes of bread odorants (Birch et al., 2014;
extraction vial the same fixed amount of a deodorised bread Pico et al., 2015): 4 alcohols, 13 aldehydes, 2 ketones, 4 furans, 4
powder sample, instead of the bread powder sample itself, and by pyrroles, 6 pyrazines, 2 other heterocyclic compounds. The evalu-
spiking the pure compounds into the extraction solution at six ation of the level of volatiles determined in the crust and in the
concentration levels. For some target analytes the calibration curve whole slice (Tables 1 and 2) showed, as expected, higher levels of
of the most structurally similar target compound was used Maillard reaction products, such as pyrazines, pyrroles and furans,
(Table 2). A triplicate analysis was performed on the bread powder in the crust than in the whole slice. The higher levels in the crust of
obtained from each loaf, for each batch. the aldehydes 2-methylpropanal, 2- and 3-methylbutanal, phe-
nylacetaldehyde and the ketone 2,3-butanedione are consistent
2.3. Sensory analysis with their formation during baking within the complex of the
Maillard reactions, as discussed below. In contrast, compounds that
Bread samples were evaluated and compared by quantitative are mainly formed during fermentation and accumulate within the
descriptive analysis (Raffo et al., 2003). A panel of professional as- crumb (2-phenylethanol, 2-/3-methyl-1-butanol) were found at
sessors with several years of experience in quantitative descriptive higher levels in whole slice samples. A two-dimensional repre-
sensory evaluation of foods and bread, in particular, analyzed the sentation of projections along the first two components resulting
bread samples. Individual bread samples, prepared as whole slices from a PCA allowed for a first exploration of the impact of the
1 cm thick, were served to the panelists in plates coded with three- considered formulation changes on the whole volatile profile of
digit random numbers at room temperature (20 ± 2  C) in an bread crust and whole slice samples (Figs. 1 and 2). In the case of
environmentally controlled sensory laboratory, provided with in- bread crust the first two PCs explained 73% of the total variance,
dividual sensory booths. The bread samples were served in with PC1 and PC2 accounting for, respectively, 49% and 24%. Most of
monadic order balanced across the panelists. The panelists agreed a volatile compounds are clearly grouped according to their route of
consensus list of 16 attributes for profiling and definitions for each formation in bread. Compounds whose main route of formation is
attribute (Table 2S). Intensity ratings were scored on a continuous linked to the Maillard reaction are grouped close to the positive axis
15 cm unstructured scale anchored at the low end (“not percep- of PC1: pyrazines, pyrroles, the Strecker aldehydes 2-
tible”, 0-value) and at the high end (“strong”, 9-value) for some methylpropanal, 2- and 3-methylbutanal, phenylacetaldehyde
descriptors, whereas distinctive anchor terms were used for others (but not methional), the ketones 2,3-butanedione and 1-octen-3-
(Table 2S). The experiment was designed so that three analytical one, some furans (2-acetylfuran and 2 furfural) and other hetero-
replicates were given to each panelist for each breadmaking cyclic compounds (2-acetylpyridine). The main products of the
replicate. Ehrlich pathway, 2-phenylethanol, 2-/3-methyl-1-butanol are
located on the opposite side, close to the negative axis of PC1.
2.4. Statistical analyses Volatiles formed through the third most important route of aroma
formation in bread, i.e. lipid oxidation (most of aldehydes and 2-
A Principal Component Analysis (PCA) was performed on the pentyl furan) are grouped close to the positive axis of PC2. A
dataset of volatile compounds determined on both bread crust and similar picture, with minor differences, is also given by the repre-
whole slice samples. Datasets used for PCA were obtained by first sentation of the PCA results obtained on the whole slice dataset,
averaging volatile levels obtained by analytical replicate de- where the first two PCs accounted for 74% of explained variance
terminations and then applying auto-scaling as pre-treatment. To (PC1 53% and PC2 22%). It is well known that some volatiles found
identify which volatiles were significantly affected by changes in in bread can be formed by more than one route of formation, which
bread formulation univariate methods of data analysis were per- sometimes complicates the interpretation of effects of processing
formed. While the assumption of normality of data on volatiles was factors: for example, the aldehydes 2-methylpropanal, 2- and 3-
confirmed, by the Shapiro-Wilk test, the assumption of homosce- methylbutanal, phenylacetaldehyde and methional can be formed
dasticity, according to the Levene test, was not. Therefore, statistical through the Ehrlich pathway during the fermentation step or can
significance of differences in volatile levels between bread formu- result from the Strecker degradation during baking (Pico et al.,
lations was evaluated by the non-parametric Kruskal-Wallis test, 2015). Similarly, 2,3-butanedione can be formed either through
whereas the Mann-Whitney test was applied to pairs of data as a the glycolysis of pyruvic acid during fermentation, or as a product of
post-hoc test of significance between pairs of results (Granato et al., the Maillard reaction occurring during baking (Pico et al., 2015),
2014). whereas furans can be derived from both lipid oxidation and the
Data from sensory analyses were checked for panel uniformity Maillard reaction (Parker, 2015a). Groupings of compounds high-
and normality. Significance of differences for data having a normal lighted by PCA representations suggest that in the conditions of the
distribution was assessed by ANOVA, and mean values for each present study the Strecker degradation, and not the Ehrlich
bread formulation were compared by a multiple comparison pathway, was likely the main route of formation of 2-
Duncan test to look for grouping. For data not having a normal methylpropanal, phenylacetaldehyde, 2- and 3-methylbutanal,
distribution significance of differences was assessed by the because in the PCA graph they are clearly associated to the other
Kruskal-Wallis test. products of the Maillard reaction and located quite far away from
All statistical analyses, except for the Mann-Whitney test, were 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 the Ehrlich pathway. In particular, results from univariate data
main biological source of 2,3-butanedione. As regards furans, 2- analysis shows that increasing NaCl level from 1.5% to 3% produced
furfural, 2-acetylfuran and 1-furfurylpyrrole were associated with a significant decrease in the level of 2-phenylethanol (23%) and a
the formation of other Maillard reaction products, whereas 2- slight (not significant) decrease of 2-/3-methyl-1-butanol (7%) in
pentylfuran, being located close to alkanals, 2-alkenals and 2,4- the whole slice (Table 2). These compounds are among the main
alkanodienals in the PCA graphs, was likely derived from oxida- components of the volatile fraction of bread crumb, are major
tion of fatty acids, as previously reported (Parker, 2015b). contributors to its yeasty note and are formed through the Ehrlich
PCA representation of data obtained on both crust and whole pathway, which is a secondary fermentation reaction carried out by
slice consistently shows that PC1 discriminated bread samples yeasts (Pico et al., 2015). Therefore, the reduction of their level in
based on their content of Maillard reaction volatile products the NaCl 3% bread can be plausibly explained by the inhibition ef-
(associated to positive PC1 values) and of compounds formed fect of NaCl on yeast activity, which has been repeatedly reported in
through the Ehrlich pathway (associated to negative PC1 values). the literature (Cauvain, 2007; He et al., 1992; Lynch et al., 2009) and
Thus PC1 effectively describes the influence of changes in formu- also observed in the present study by measurements of gas pro-
lation on these two routes of formation of volatiles. When duction during dough fermentation (data not reported). In previous
comparing breads with NaCl, it is clear that a higher level of added studies, reduced levels of gas production in the dough, and thus of
salt promoted the progress of the Maillard reaction and inhibited 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., of the Maillard reaction. One possible explanation was that
2009; Pflaum et al., 2013), were observed in bread with increased increased levels of NaCl can promote the formation of melanoidins
NaCl levels. In the present study, the observation of a reduction in by the Maillard reaction (Silow et al., 2016), an effect that resembles
the level of 2-phenylethanol, while confirming recently reported that one observed in the present study on the volatile products of
similar results (Belz et al., 2017), provides a further insight into the the Maillard reaction. NaCl addition may increase the rate of the
molecular basis of the reduced perception of the yeasty note. Maillard reaction through several mechanisms: by influencing
Another effect of increasing NaCl level from 1.5% to 3% is the water activity, moisture content and pH of the dough, or by a
enhanced formation in the crust of most of the Maillard products: plasticising effect on the food matrix during the backing step,
the key odorants pyrazines (3-ethyl-2,5-dimethylpyrazine, 2-ethyl- which could improve the mobility of the reactants of the Maillard
3,5-dimethylpyrazine), the Strecker aldehydes (2-methylpropanal, reaction, thus favouring the progress of the reaction (Moreau et al.,
2- and 3-methylbutanal, phenylacetaldehyde), some furans (2- 2009). Alternatively, it has been suggested that the lowered yeast
furfural, 2-acetylfuran), and the pyrroles (1-furfurylpyrrole, 2- activity observed during dough fermentation and associated to the
acetylpyrrole, 2-fromylpyrrole) (Tables 1 and 2). Increased levels addition of NaCl, could leave an increased amount of sugars not
of NaCl in the dough have been observed to promote crust colour metabolised at the end of fermentation and thus an increased pool
formation in bread and breakfast cereals (Moreau et al., 2009; Silow of them could be available for the formation of browning Maillard
et al., 2016) and therefore have been supposed to increase the rate 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 ethyl-3,5-dimethylpyrazine, 2-acetylfuran, 2-formylpyrrole), and
volatile Maillard products. Only a direct measurement of an effect only at the 1.5% addition level. The presence of lysine in the
on sugar levels in the dough at the end of the fermentation step formulation of Pansalt®, corresponding to a concentration of 1.6
would support the hypothesis that the enhanced level of Maillard and 3.3 mmol of lysine per Kilogram of flour, for addition of Pan-
volatiles could have been due to a NaCl effect on the yeasts activity salt® at 1.5 and 3%, respectively, could in principle favour the for-
and thus on the concentration of precursors of the Maillard mation of Maillard products with respect to the formulations with
reaction. NaCl. However, the presence of an effect only at the lower addition
PCA results show that a similar influence on the Ehrlich pathway level but not at 3%, suggested that the presence of lysine in the salt
and the Maillard reaction products was also observed when replacer was not likely the factor that could explain the increased
comparing the two breads with Pansalt®, even though the effect of formation of Maillard compounds in bread with Pansalt® at 1.5%.
the increased addition level was less marked (Figs. 1 and 2). A Considering the second Principal Component in the PCA graph,
significant reduction of the level of 2-phenylethanol, but not of 2-/ it can be seen that it discriminates bread samples based on their
3-methyl-1-butanol, and an increase of many volatile Maillard re- level of lipid oxidation products (associated to positive PC2 values),
action products in the crust was also observed, even though the highlighting a lower level of this group of compounds in the bread
impact was generally less marked than in the case of NaCl (Tables 1 with 1.5% Pansalt® with respect to all the other breads. This effect
and 2). seems not to be easily explainable based on experimental data
As regards the effect of substitution of NaCl with Pansalt®, the collected in the present study.
PCA representation suggests that at 1.5% it tended to favour the PCA representations also allowed for a visual evaluation of the
formation of Maillard reaction products and to inhibit the Ehrlich extent of variation of the volatile profile associated to the experi-
pathway, whereas at the level of 3% this effect was less pronounced mental procedure for breadmaking, as measured by the distance in
(Figs. 1 and 2). Univariate analysis shows that at both levels of the graph between the two replicates for each bread formulation.
addition, substitution of NaCl by Pansalt® is accompanied by a
significant reduction of 2-phenylethanol and 2-/3-methyl-1- 3.2. Effects of different levels of NaCl and Pansalt® on bread sensory
butanol levels (Tables 1 and 2), but it is not possible to state here profile
whether this effect was mediated by an influence of the different
inorganic salts composition on yeasts activity. In contrast, the Besides the obvious more intense perception of saltiness,
substitution of NaCl by Pansalt®, promoted the formation of only significant changes in other sensory attributes were produced by
few of the Maillard products (3-ethyl-2,5-dimethylpyrazine, 2- 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 525

was associated with a less intense yeasty note, as perceived on 4. Conclusions


the crumb. A similar effect was previously observed on wheat
bread by increasing salt level from 0 to 2%, based on flour In this study for the first time the influence of NaCl addition on
(Pflaum et al., 2013), and it is consistent with effects of NaCl the formation of bread aroma compounds was investigated in
increase on formation of 2-phenylethanol and 2-/3-methyl-1- detail, highlighting a significant impact of the addition level on
butanol found in the present study (Section 3.1). A higher level some of the most important routes of formation of volatiles.
of NaCl was also accompanied by more intense toasted flavour Increasing NaCl level from 1.5 to 3% gave rise to a significant
and aroma, as perceived on the crust. Also this effect was reduction of the yeasty smelling crumb odorant 2-phenylethanol,
consistent with results of volatile profiling, the more intense while promoting the formation of many volatile products of the
toasted notes corresponding to the higher concentration of the Maillard reaction. In addition, significant changes in several sen-
Maillard volatile products observed in the 3% NaCl bread sory attributes besides saltiness, such as aroma, other taste and
(Table 1). The increase of toasted notes perceived on bread crust texture attributes were observed. Some of these additional effects
paralleled an even more marked increase of two other taste can be related to the influence of NaCl level on yeast activity during
sensations, also perceived on the crust, such as bitter taste and fermentation, on gluten physical properties and reactions occurring
aftertaste. An opposite effect of NaCl on bitterness was previously during baking. Moreover, data were provided on the effects of the
observed, but in that case the sensory evaluation was performed use of a commercial salt substitute on volatiles formation and
on crumb instead of crust (Pflaum et al., 2013). In another study sensory quality. Substitution of NaCl by the mixture of inorganic
no effect was observed on bitterness, but it was not specified salts and taste enhancers present in Pansalt® was only partially
whether crust or crumb samples were analyzed (Lynch et al., effective in maintaining the saltiness elicited by NaCl, while giving
2009). Whether the increase in the bitter sensations observed rise to perceptible increase in bitter taste and aftertaste in crust at
in the present study could be due to higher levels of non volatile the 3% addition level.
products of the Maillard reaction could be the object of further
investigations. The crumb of bread with 3% NaCl was also Funding
perceived as less sweet than the 1.5% counterpart. In contrast,
salt increase has been sometimes reported in the past to have a This work was supported by the Italian Ministry of Agriculture
promoting effect on bread sweetness (Miller and Hoseney, 2008), and Forestry [EUSAL Project].
whereas in recent experimental studies no effect of NaCl increase
was observed on sweetness (Lynch et al., 2009; Pflaum et al., Conflicts of interest
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 authors declare no conflict of interest.
the formulation used in the present study (0.36%) could have
levelled off the perception of sweetness. As regards the effects on Acknowledgment
bread texture, the results reported in the present study
confirmed that an increase in NaCl level gave rise to a more even Thanks are due to Mr. L. Bartoli for technical help in bread
crumb structure, with smaller and more homogeneous pores. making.
This effect had been previously observed and explained on the
basis of the influence of NaCl addition on gluten physical prop- Appendix A. Supplementary data
erties and the resulting enhanced stability of water vapor bub-
bles during baking (Lynch et al., 2009; Silow et al., 2016). In Supplementary data (Tables 1S and 2S) associated with this
addition, in the present study a slight (not significant) increase article can be found in the online version, at https://doi.org/10.
was also observed at the 3% level on crumb elasticity and crust 1016/j.jcs.2017.12.013.
crispness.
Increasing the addition level of Pansalt® from 1.5 to 3% had a
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