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Articol Anca J Cereal Sci
Articol Anca J Cereal Sci
Insights into the fermentation process of fresh and frozen dough bread
made with alginate-immobilized S. cerevisiae yeast cells
L. Mihaly Cozmuta a, *, C. Nicula a, A. Peter a, R. Apjok a, A. Jastrzębska b, A. Mihaly Cozmuta a
a
Technical University of Cluj-Napoca, Department of Chemistry-Biology, 76 Victoriei Str, Baia Mare, Romania
b
Warsaw University of Technology, Faculty of Materials Science and Engineering, Woloska 141, 02-507, Warsaw, Poland
A R T I C L E I N F O A B S T R A C T
Keywords: The study reveals the influence of ascorbic acid and salt on the fermentation of fresh and frozen doughs made
S. cerevisiae encapsulation with free and alginate-immobilized yeast cells. The evolution profiles of Saccharomyces cerevisiae, the redox
Frozen dough bread potential, pH, and specific volume were monitored and the most suitable conditions that resulted in bread loaves
Dough redox potential
being highly accepted by consumers were selected. For fresh doughs, the maximum specific volumes were
Dough-specific volume
recorded for the dough made with free yeast cells (DYD-3.0), 6 g NaCl/kg of flour, and 0.5 g ascorbic acid/kg of
flour, combining a negative redox potential (− 3 mV), a pH of 4.07, and 2 h of proofing. The highest specific
volume for the 3-months freezing dough with immobilized yeast cells (CAYD-1.3) requires 6 g NaCl/kg of flour,
1 g ascorbic acid/kg of flour, and combines a negative redox potential (− 2 mV), a pH of 3.82, and 2 h of proofing.
The bread loaf from CAYD-1.3 received the highest average score for the overall acceptability which is higher
than that given to the bread baked from fresh dough DYD-3.0.
* Corresponding author.
E-mail address: mihalyleonard@yahoo.com (L. Mihaly Cozmuta).
https://doi.org/10.1016/j.jcs.2022.103516
Received 1 March 2022; Received in revised form 23 May 2022; Accepted 12 June 2022
Available online 20 June 2022
0733-5210/© 2022 Elsevier Ltd. All rights reserved.
L. Mihaly Cozmuta et al. Journal of Cereal Science 107 (2022) 103516
Table 1
Formulas of dough pieces used in the experimental design.
Dough Thermal status of the White Dry yeast, g Amount of beads, g Salt, g Ascorbic acid, g Water, mL
code dough flour,
g (g/Kg of flour) (g/Kg of flour) (g/Kg of flour) (g/Kg of flour) (mL/Kg of flour)
Formula 1
DYD-1.0 Fresh 500 5 (10 g/Kg of – 3 (6 g/Kg of flour) 0.5 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour)
CAYD-1.0 500 – 23.5 (47 g/Kg of 3 (6 g/Kg of flour) 0.5 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour)
DYD-1.3 3-months frozen 500 5 (10 g/Kg of – 3 (6 g/Kg of flour) 0.5 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour)
CAYD-1.3 500 – 23.5 (47 g/Kg of 3 (6 g/Kg of flour) 0.5 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour)
Formula 2
DYD-2.0 Fresh 500 5 (10 g/Kg of – 6 (12 g/Kg of 0.25 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour) flour)
CAYD-2.0 500 – 23.5 (47 g/Kg of 6 (12 g/Kg of 0.25 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour) flour)
DYD-2.3 3-months frozen 500 5 (10 g/Kg of – 6 (12 g/Kg of 0.25 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour) flour)
CAYD-2.3 500 – 23.5 (47 g/Kg of 6 (12 g/Kg of 0.25 (1 g/Kg of flour) 350 (700 mL/Kg of
flour) flour) flour)
DYD -3.0 Fresh 500 5 (10 g/Kg of – 3 (6 g/Kg of flour) 0.25 (0.5 g/Kg of 350 (700 mL/Kg of
flour) flour) flour)
CAYD-3.0 500 – 23.5 (47 g/Kg) 3 (6 g/Kg of flour) 0.25 (0.5 g/Kg of 350 (700 mL/Kg of
flour) flour)
DYD -3.3 3-months frozen 500 5 (10 g/Kg of – 3 (6 g/Kg of flour) 0.25 (0.5 g/Kg of 350 (700 mL/Kg of
flour) flour) flour)
CAYD-3.3 500 – 23.5 (47 g/Kg of 3 (6 g/Kg of flour) 0.25 (0.5 g/Kg of 350 (700 mL/Kg of
flour) flour) flour)
The dough pieces were assigned a code consisting of an abbreviation followed by two numbers: the abbreviations DYD and CAYD were assigned to the dough pieces
made with free and immobilized yeast cells, respectively; the first number in the code indicates the formula with which the dough was prepared, while the second
number indicates whether the dough was fresh (0) or frozen for 3 months (3).
DYD -1.0; DYD-2.0; DYD-3.0 – fresh dough pieces containing free yeast cells, prepared according to formulas 1, 2 and 3, respectively.
DYD -1.3; DYD-2.3; DYD-3.3 – 3-months frozen dough pieces containing free yeast cells, prepared according to formulas 1, 2 and 3, respectively.
CAYD-1.0; CAYD-2.0; CAYD-3.0 – fresh dough pieces with immobilized yeast cells, prepared according to formulas 1, 2 and 3, respectively.
CAYD-1.3; CAYD-2.3; CAYD-3.3 – 3-months frozen dough pieces with immobilized yeast cells, prepared according to formulas 1, 2 and 3, respectively.
et al., 2020; Noort et al., 2012) or the sensorial characteristics of the associated with the fresh and frozen dough bread that was the most
bread (Bryszewska et al., 2019; Ezhilarasi et al., 2013; Pasrija et al., appreciated by the consumers.
2015; Zhang et al., 2018). The capsules could influence the fermentation
stage by interfering with the physical, chemical, and microbiological 2. Materials and methods
processes occurring in the dough. The encapsulated bioactive com
pounds alter the redox balance of the dough and may result in bread 2.1. S. cerevisiae yeast cell immobilization
with poor sensorial characteristics due to their oxidizing (some metallic
ions such as Fe3+) or reducing (vitamins, carotenoids, flavonoids, and Beads made of dry yeast cells (DY) immobilized in an alginate matrix
Fe2+) potential (Alwazeer, 2020). An improved understanding of the with the characteristics described by Mihaly Cozmuta et al. (2021) were
fermentation of such dough could help select the optimal parameters to used in the experiment. Briefly, the beads (CAY) were prepared from
obtain bread with sensorial characteristics highly accepted by alginate:yeast:water in a ratio of 2:5:100 (w:w:v) with an encapsulation
consumers. efficiency of 72.79% and carried a yeast cell load of 1.38 × 10− 7 CFU/g.
Our previous work (Mihaly Cozmuta et al., 2021) investigated the The equivalence between CAY beads and DY in terms of yeast cell load
immobilization of S. cerevisiae baker’s yeast cells in an alginate matrix in was 4.70 g of CAY beads/1 g DY (dry matter basis).
order to protect them during the freezing – frozen storage – thawing
process and improve the sensorial characteristics of bread produced 2.2. Frozen dough preparation and storage
from frozen dough. The 3-month frozen dough bread made with yeasts
immobilized in a calcium alginate matrix (CAYD) collected high, but not Three dough formulas were investigated, as depicted in Table 1. The
maximum, average scores for crust color, firmness, taste, smell, and reference formula (formula 3) described the ingredients in the ratios that
overall acceptability. led to the fresh and 3-month frozen dough loaves that received the
In this context, this paper aims to identify and select the conditions highest sensory scores, as described in our previous work (Mihaly Coz
that improve the above-mentioned sensorial attributes. The paper is muta et al., 2021). The amount of yeast (Pakmaya, Romania) followed
organized as follows: (i) constructing operating diagrams based on the the manufacturer’s recommendation, and the amount of beads added
evolution of the S. cerevisiae count, redox potential, pH, and specific was equivalent to the amount of yeast (4.70 g of CAY beads/1 g DY).
volume during fermentation, and the values associated with the highest Another two dough types (formula 1 and formula 2) were prepared by
specific volumes of doughs were selected; (ii) baking loaves from the varying the ratios of salt (Salrom, Romania) or ascorbic acid (AsA,
investigated doughs with the selected parameters, which were subjected Merck, Germany) (Table 1). Preliminary investigations were conducted
to sensorial study; and (iii) determining the fermentation parameters by adding more or less ascorbic acid and salt than those given in Table 1,
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L. Mihaly Cozmuta et al. Journal of Cereal Science 107 (2022) 103516
Fig. 1. Evolution profiles of redox potential, S. cerevisiae growth, pH and specific volume associated to fermentation of fresh and 3-months frozen dough pieces made
with free yeast cells:
a) DYD-1.0; b) DYD-2.0; c) DYD-3.0; d) DYD-1.3; e) DYD-2.3; f) DYD-3.3. DYD -1.0; DYD-2.0; DYD-3.0 – fresh dough pieces containing free yeast cells, prepared
according to formulas 1, 2 and 3, respectively;
DYD -1.3; DYD-2.3; DYD-3.3 – 3-months frozen dough pieces containing free yeast cells, prepared according to formulas 1, 2 and 3, respectively.
which led to severe failures in terms of the physical and sensory char sampling was conducted to determine the specific volume and yeast cell
acteristics of the resulting bread. count.
After mixing the ingredients for 10 min in a laboratory dough mixer
(Bilancia, Romania), the dough pieces were wrapped in plastic foil and
frozen for 3 months at − 18 ◦ C (Zanussi Chest Freezer, Pordenone, Italy), 2.3. S. cerevisiae yeast cell counts
at an average freezing rate of 3.2 ◦ C/min. Upon removal, the frozen
dough was allowed to thaw for 2 h, until the dough pieces reached 22 ◦ C, The viable yeast cells in the dough samples were counted according
to activate the yeast, then divided into smaller parts and kept for 6 h at to ISO 6887–4:2017. Samples of dough (5 g) were gently homogenized
32 ◦ C and 75% relative humidity in a proofing chamber (Matina D 6040, in 45 mL of sterile buffered peptone water (0.1% w/w) in a stomacher,
Bucharest, Romania). and 0.1 mL from each serial dilution of suspension up to 10− 5 was
The pH and redox potential of fresh and thawed dough pieces were transferred onto Petri dishes with DRBC Agar (Dichloran Rose Bengal
continuously monitored during fermentation. Simultaneously, periodic Chloramphenicol Agar, Oxoid, LTD, England). The plates were incu
bated for 5 days at 25 ◦ C, and the colonies that developed were counted.
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L. Mihaly Cozmuta et al. Journal of Cereal Science 107 (2022) 103516
Fig. 2. Evolution profiles of redox potential, S. cerevisiae growth, pH and specific volume associated to fermentation of fresh and 3-months frozen dough pieces made
with immobilized yeast cells:
a) CAYD-1.0; b) CAYD-2.0; c) CAYD-3.0; d) CAYD-1.3; e) CAYD-2.3; f) CAYD-3.3. CAYD-1.0; CAYD-2.0; CAYD-3.0 – fresh dough pieces with immobilized yeast cells,
prepared according to formulas 1, 2 and 3, respectively;
CAYD-1.3; CAYD-2.3; CAYD-3.3 – 3-months frozen dough pieces with immobilized yeast cells, prepared according to formulas 1, 2 and 3, respectively.
2.4. pH and redox potential measurement 2.5. Specific volumes of dough pieces and bread loaves
The pH was measured with a pH electrode (WTW InoLab, pH 730, The specific volume of dough pieces during fermentation and the
Germany) previously calibrated at two points in pH 7 and pH 4 buffer final volume of the corresponding bread loaves were measured ac
solutions. The redox potential (related to the standard hydrogen elec cording to the rapeseed displacement method (Mihaly Cozmuta et al.,
trode, Eh) was measured with a redox electrode (HANNA Instruments, 2015).
pH/ORP/oC meter, HI991003, Italy). The pH and Eh electrodes were
punched directly into the dough sample and remained in place
2.6. Bread baking and sensorial study
throughout the experiment to avoid technical problems (signal errors,
air bubbles).
Based on the variation profile of the specific volumes, the proofing
time required for each dough type to achieve the maximum specific
volume was determined and used to bake the bread. The fresh and
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thawed 810-g dough pieces were prepared according to Table 1, shaped lag phase. The fresh doughs made with encapsulated yeast cells
into a round form, proofed, and baked (details are presented in the (CAYD-1.0, CAYD-2.0, CAYD-3.0) displayed longer lag phases compared
Supplementary Material). The bread loaves were marked with the codes to the corresponding doughs made with free yeast cells. These length
assigned to the dough pieces they originated from (Table 1) by replacing ened lag phases seem to result from the leakage rate of cells embedded in
the letter “D” (dough) with “B” (bread). The resulting bread loaves were the alginate beads into the dough rather than the yeast adjustment to the
subjected to a sensorial study according to the protocols detailed in substrate. Since the alginate is a pH-responsive polymer, it swells and
Section 2.1. SM from Supplementary Material. The attributes of crust even dissolves in an environment containing anions with calcium af
color and firmness, crumb aspect, bread volume, smell, taste, and overall finity. The calcium ions from the alginate matrix leach out in an acidic
acceptability were evaluated using a five-point hedonic scale (1 – dislike environment, leaving cavities that can be pathways to release the
extremely to 5 – like extremely). The overall sensory score of each embedded yeast cells (Han et al., 2007). The cracks and voids that
attribute was computed as the average of the individual scores provided appeared on the surface of beads during the freezing – frozen storage –
by the panelists. thawing process (Mihaly Cozmuta et al., 2021) favored the diffusion of
yeast cells from beads into the dough and shortened the lag phase. An
2.7. Statistical analysis increase in the ionic strength in CAYD-2.0 due to the excess NaCl and the
preference of alginate for Ca2+ rather than Na + limited the solubility of
All investigations were performed in triplicate, and the results were the alginate beads and restricted the leakage of yeast cells, resulting in a
expressed as mean ± standard deviation. One-way analysis of variance more extended lag phase of about 60 min.
(ANOVA) and Student’s t-test were used to assess the difference between After the yeast cells exit the lag phase, they undergo growth and
the two means. The probability value p ˂ 0.05 was considered statisti multiplication following a curve whose shape approximates an expo
cally significant. nential equation (log phase) at different rates, depending on the pre
vailing conditions and the number of accumulated metabolites. After
3. Results and discussions they become active, the yeast cells quickly convert glucose and consume
oxygen through respiration to generate ATP for growth. Thus the pop
Figs. 1 and 2 illustrate the influence of salt and ascorbic acid on the ulation of viable cells is characterized by a low fermentation efficiency
growth of S. cerevisiae cells in fresh and frozen doughs, as well as the (Liu et al., 2011). As oxygen depletion occurs, the metabolism of yeast
redox potential, pH, and specific volume of the dough mentioned above cells modulates from aerobic growth to anaerobic glucose fermentation
types. The standard deviations ranged between 0.04 and 0.19 for the to ethanol and secondary metabolites, whose CO2 is essential in the
yeast cell count, 0.03–0.14 for the pH, 2.65–6.93 for the redox potential, bread-making process. In the fresh doughs, the lowest growth rate of
and 0.08–1.30 for the specific volume. Features of the yeast cells growth, 8.48% occurred in the DYD-2.0 (Table 3SM) in response to the osmotic
redox potential, pH and specific volume profiles in the investigated stress induced by the high concentration of salt (1.2%), which caused
dough types extracted from Figs. 1 and 2 are shown in Table 3SM, 4. SM, the plasmolysis of cells and retarded their multiplication. Bonjean and
5. SM and 6. SM from the Supplementary Material. Guillaume (2003) have also reported reduced fermentation at a salt
content above 1%. As expected, the highest multiplication rate was
3.1. Biomass growth profiles observed for the dough with free yeast cells DYD-1.0 (32.42%) because
the larger amount of ascorbic acid in this dough stimulated the yeast
The growth profiles of S. cerevisiae cells revealed four distinctive growth (Echevarria et al., 2010) to a larger extent than in DYD-3.0
phases: lag, log, stationary, and decline (Figs. 1 and 2). In the lag phase, (24.94%), in which the amount of AsA is two times smaller. Interme
the free yeast cells try to acclimate to the substrate, but some of them do diate values of 29.03%, 11.29%, and 29.57% were found for the growth
not adjust successfully, and the number of cells that have yet to begin to rates of yeast cells in the CAYD-1.0, CAYD-2.0, and CAYD-3.0 fresh
divide is almost equal to the number that have died. Of all fresh doughs doughs, respectively, as the immobilization of the yeast constrained
made with free yeast cells, the shortest lag phase, around 20 min, was their access to the substrate and therefore retarded growth. As seen in
observed in DYD-1.0 and DYD-3.0 (Table 3SM.). The brevity of their lag Figs. 1 and 2, the doughs subjected to frozen storage for 3 months dis
phase indicated that the yeast cells contain the full complement of played higher yeast cell growth rates compared to the fresh doughs. This
fermentative enzymes (Jackson, 2020), and the switch from respiratory can be accounted for by the lower density of yeast cells competing for
to fermentative metabolism required only a short amount of time. the substrate nutrients. In addition, in the case of the doughs made with
Conditions such as NaCl presence and low pH may disadvantage yeast immobilized yeast, the corrosion of the bead walls that occurred during
cells, as a result of the osmotic stress they experience (Wei et al., 1982; the freezing – frozen storage – thawing process favored the faster release
Jackson, 2020). Although the NaCl and AsA are present simultaneously, of the yeast into the dough. The highest growth rate, 49.11%, occurred
a rapid transition of yeast cells to the log phase can be observed for in CAYD-1.3, the thawed dough that contained immobilized cells, while
DYD-1.0 and DYD-3.0, respectively. The small amounts in which they in DYD-1.3, the corresponding dough made with free yeast, the growth
were added seems to have a low negative impact on yeast cells, resulting rate was 33.06%. The stress that occurred during the freezing – frozen
in short lag phase. storage – thawing process affected the free yeast cells to a larger extent
The elevated salt level in DYD-2.0 extended the lag phase to around than the immobilized ones, not only in terms of their viability but also
40 min, showing the difficulties of yeast adapting to the osmotic stress. the quality of the surviving cells, given the reported mutagenic effect on
The accumulation of trehalose and glycerol is a part of the yeast’s their mitochondrial DNA (Stoycheva et al., 2007). As outlined in
strategy to maintain the plasma membrane and stabilize the structural Table 3SM, a time range of 120–300 min is required to achieve the
proteins (Eardley and Timson, 2020; Jackson, 2020). The prolongation highest growth rates of free and immobilized cells in fresh and thawed
of the lag phase was also observed for the doughs made with free yeast doughs.
cells subjected to the freezing – frozen storage – thawing process The lack of nutrients accompanied by the accumulation of toxic
(DYD-1.3, DYD-2.3, DYD-3.3). This effect is possibly due to the yeast metabolic by-products, such as mid-chain carboxylic acids (C8 and C10)
populations containing young and mature cells that were differently and their esters (Jackson, 2020), led to the stationary phase (Figs. 1 and
affected by frozen storage. Old cells may handle stress more efficiently 2), which is characterized by arrested growth, although the fermenta
than young ones (Lippuner et al., 2014), as the time required for re tion process still occurs. The cells maintain their viability without added
covery after thermal stress is longer for young cells, as part of their nutrients, depending on their physiological state. The yeast cells in
energy is oriented toward repair mechanisms rather than growth (Smart, DYD-1.0, which were not stressed by high salinity, immobilization, or
2001). All these processes are directly correlated with the length of the the freezing – frozen storage – thawing process, extended their viability
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during the stationary phase by up to 80 min (Table 3SM). Shorter sta associated with the stationary phase of the yeast growth curve.
tionary phases of around 60 min are associated with the cells restrained Depending on the composition of the dough, it can continue with an
in the alginate matrix, subjected to freezing – frozen storage – thawing uprising branch or be maintained during the rest of the experiment.
process, or developed in acidic doughs. Similar to what was observed in The uprising branch of the redox profile can be associated with the
the growth phase, an increased salt level reduced the length of the sta decline phase in the growth profile of yeast cells. Of all the redox pro
tionary phase in DYD-2.0 to 40 min, while the combination between files, this behavior is displayed only by DYD-1.0, DYD-1.3, DYD-3.0, and
salinity and freezing – 3-month frozen storage – thawing reduced it even DYD-3.3 (Fig. 1a, d, 1c, 1f), indicating slowed or stalled fermentation as
more, to 20 min in DYD-2.3. The protection of immobilized cells against a result of yeast cell lysis and resulting accumulation of oxidative me
high salinity and freezing exerted by the alginate matrix is reflected by tabolites in the dough (Liu et al., 2013). The concordance between the
stationary phases of around 40 min observed in CAYD-2.0 and redox profile and the yeast cell growth profile in DYD-1.0 and DYD-3.0 is
CAYD-2.3. seen in the good alignment of the basins of the redox profiles and the
With the gradual accumulation of toxic metabolites (octanoic and stationary phases in the growth profiles. The appearance of the uprising
decanoic acids and their esters) and the reduction of nutrients, more branches in the doughs mentioned above can be correlated with severe
yeast cells die or become dormant than those that divide (Jackson, drops in the decline phases of the yeast cell growth curves (Figs. 1 and
2020), and the biomass enters the decline phase (Figs. 1 and 2). Three 2), which indicate the accumulation of large amounts of oxidative
patterns can be observed in the decline phase: (1) it was not achieved compounds. The redox profiles of fresh and thawed doughs made with
during the investigated time range in DYD-1.0, DYD-1.3, CAYD-1.0, and immobilized yeasts not subjected to NaCl stress maintain their basins.
CAYD-3.3; (2) there was a smooth decline in DYD-1.3, DYD-3.3, This can be explained by the absence (CAYD-1.0, CAYD-1.3, and
CAYD-3.0, and CAYD-1.3; and (3) an abrupt decline occurred in CAYD-3.0) or low values of the decrease rate of yeast cells (CAYD-3.3).
DYD-2.0, DYD-2.3, CAYD-2.0, and CAYD-2.3. The doughs displayed different reduction capacities in terms of
minimal Eh values (Table 4SM). The redox potential in the fresh doughs
3.2. Redox potential profiles prepared with free yeast cells constantly decreased to negative values.
The lowest values, − 38 and − 37 mV, were recorded in DYD-3.0 and
The redox profiles (Figs. 1 and 2) of the investigated doughs followed DYD-2.0, compared to − 4 mV in DYD-1.0. In the corresponding thawed
either a bathtub curve (DYD-1.0, DYD-1.3, DYD-3.0, DYD-3.3), a doughs, the minimum Eh became less reduced, and higher minimal
continuously declining curve (DYD-2.0, DYD-2.3, CAYD-2.0, CAYD-2.3), values were reached: − 27, 62, and 75 mV. By comparison, the minimum
or a declining curve followed by a plateau (CAYD-1.0, CAYD-3.0, CAYD- Eh in the fresh dough made with immobilized yeast cells (CAYD-1.0,
1.3, CAYD-3.3). All initial redox potentials are above 100 mV CAYD-3.0) had positive values. Furthermore, positive values dropped to
(Table 4SM). High values were associated with the lag phase, and log negative ones in the thawed doughs CAYD-1.3 and CAYD-3.3, indicating
arithmic behavior at the onset of the lag phase was due to oxygen an increase in the reduction power of the dough. This effect is related to
incorporated into the dough during mixing. The oxygen triggered the the highest growth rates in the study, which occurred in CAYD-1.3 and
respiratory circuit and resulted in low fermentation efficiency (Liu et al., CAYD-3.3 (Table 4SM). The behavior of the minimal Eh in CAYD-2.3
2011). The development of biomass in a high redox potential environ resembled that of free yeast doughs; its value (67 mV) was higher
ment favors the synthesis of unsaturated fatty acids in cells that will be than that in the fresh dough CAYD-2.0 (27 mV).
used to build a well-structured and highly protective cell membrane (Liu
et al., 2011). 3.3. pH evolution profiles
As the log phase developed, the electrons captured by the oxygen
dissolved in the dough could not compensate for the electrons released Acidification fulfills important functions in bread in terms of the
via biomass formation (Lin et al., 2010), as the environment became modulation of dough rheological properties, flavor, and texture, passing
more reduced and the redox potential declined until it plateaued. In the through a range of pH values close to the optimum for various enzymes
absence of oxidizing compounds in the dough, the redox potential could present in the dough that result in structural changes, retardation of
drop to negative values. The work of Capuani et al. (2014) correlated the stalling, prevention of bread aging, and delay to bacterial spoilage
negative slope of the dough redox profile with the initial yeast cell load, (Komlenić et al., 2012).
as the low-inoculation dough showed a delay in the reduction step, Fig. 1 shows continuously decreasing pH values of the fresh doughs
reduction rate, and minimal reduction value. In our study, the reduction prepared with free yeast cells. The early pH curves show a small decline,
rate depended significantly on the initial cell load and stress factors that aligned to the lag phase of the corresponding yeast cells’ cells’ growth
could inhibit or delay their development. Even if they had almost the profiles. Thus, in DYD-1.0, the biomass grew 0.2% in the first 20 min and
same initial cell load of 4.01 × 105 CFU/g, DYD-1.0, DYD-2.0, and another 2.53% over the following 20 min, while the pH decreased by
DYD-3.0 displayed reduction rates of 0.78, 0.50, and 0.67 mV/min, 0.45% and 2.40%, respectively. To a lesser extent, the same trend was
respectively (Table 4SM). The sharpest decline, observed in DYD-1.0, also observed in CAYD-2.0 and CAYD-3.0. Only a small part of the
can be attributed to a more favorable growth environment for the fermentable sugars was fermented in the lag phase. The number of acidic
yeast cells than in DYD-3.0 and DYD-2.0. In the corresponding frozen metabolites (such as succinic acid and acetic acid) released into the
doughs, lower reduction rates were calculated as 0.52 mV/min in dough was reduced, and the pH declined slightly. A good alignment can
DYD-1.3, 0.29 mV/min in DYD-2.3, and 0.65 mV/min in DYD-3.3. The be observed in DYD-1.0 and DYD-3.0 between the biomass growth, Eh,
reduction process was delayed in the doughs made with immobilized and pH profiles: the exponential phases correspond to the rapid decrease
yeast cells and reflected by the reduction rates, which were below those in Eh and pH, while the beginnings of the stationary phases were syn
made with free yeast cells. The reduction process occurs in CAYD-1.3 at chronized with the Eh and pH basins. Regarding the thawed doughs with
a higher rate (0.54 mV/min) than in the corresponding DYD-1.3 (0.52 free yeast cells, the pH declined at higher rates than in the counterpart
mV/min), correlated with the growth rates (49.11% and 33.06%, fresh doughs, while lower values of the minimum pH were achieved in a
respectively) rather than with the initial yeast cell load (1.12 × 105 shorter time (Table 5SM). This indicated more intense fermentation due
CFU/g and 1.21 × 105 CFU/g). A particular case is the doughs that to the decrease of the yeast cell load and easier access to the substrate.
display a continuous decrease of the reduction potential (DYD-2.0, Doughs made with immobilized yeasts in an alginate matrix had
DYD-2.3, CAYD-2.0, and CAYD-2.3). The life cycle of yeast expressed as interesting pH profiles (Fig. 2) due to two parallel processes that
long lag phases, low growth and reduction rates, and short stationary released products with opposite chemical properties. On the one hand,
phases is probably responsible for the decrease. the yeast cells on the surface of the beads detached easily, adjusted to the
The basin of the redox profile (i.e., the plateau of the curve) can be dough, grew, and initiated the fermentation process with the release of
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sensorial study are depicted in Fig. 4. The sensorial study is detailed in prolonged fermentative activity, enhanced gas production, and conse
the section 3.6. SM from Supplementary Material, aspects related to the quently, in the larger volumes of CAYB-1.3 and CAYB-3.3.
final volumes of bread loaves being discussed in this section. Different Comparing the average scores given to the CAYB-1.3 bread in the
trends were observed when the final volumes of bread loaves were present study (Fig. 4b) and the CAYB-3.3 bread in our previous study
compared to the highest specific volumes of the doughs from which they (Mihaly Cozmuta et al., 2021) indicated that doubling the amount of
originated. The following series resulted from the average scores given AsA in the dough formula resulted in improved scores for crust color,
by the panelists to the volume of the fresh bread loaves (Fig. 4a): crumb texture, volume, smell, and overall acceptability, while crust
firmness and taste received the same high scores.
DYB-3.0 > DYB-1.0 > CAYB-3.0 > DYB-2.0 = CAYB-1.0 > CAYB-2.0,
9
L. Mihaly Cozmuta et al. Journal of Cereal Science 107 (2022) 103516
redox potential (− 2 mV), a more acidic pH (3.82), and a shorter proofing Capuani, A., Behr, J., Vogel, R.F., 2013. Influence of lactic acid bacteria on redox status
and on proteolytic activity of buckwheat (Fagopyrum esculentum Moench)
time (2 h).
sourdoughs. Int. J. Food Microbiol. 165, 148–155.
In terms of sensory attributes, doubling the amount of salt above the Capuani, A., Behr, J., Vogel, R.F., 2014. ORP Measurements as a Monitoring Tool in
reference value negatively affected both fresh and frozen doughs. The Sourdough Fermentations. Baking+biscuit, p. 62. ISSU E 01. 2014.
saline stress altered the viability of the yeast cells, disrupted the gluten Chen, G., Ehmke, L., Sharma, C., Miller, R., Faa, P., Smith, G., Li, Y., 2019.
Physicochemical properties and gluten structures of hard wheat flourdoughs as
network, and resulted in bread with poor sensory characteristics. In affected by salt. Food Chem. 275 (1), 569–576.
comparison, doubling the amount of ascorbic acid above the reference Collins, B., Haley, S., 1992. Frozen bread doughs: effect of ascorbic acid addition and
value particularly favored the frozen dough made with immobilized dough mixing temperature on loaf properties. Chorleywood Digest 114, 21–23.
Eardley, J., Timson, D.J., 2020. Yeast cellular stress: impacts on bioethanol production.
cells (CAYD-1.3), resulting in bread loaves with sensorial characteristics Fermentation 6 (4), 109.
that scored higher than those made with free yeast cells and were highly Echevarria, R., Bautista-Gallego, J., Arroyo-López, F.N., Garrido-Fernández, A., 2010.
accepted by the consumers. Modelling the effect of ascorbic acid, sodium metabisulphite and sodium chloride
onthe kinetic responses of lactic acid bacteria and yeasts in table olive storage using
a specifically implemented Quasi-chemical primary model. Int. J. Food Microbiol.
CRediT author statement 138, 212–222.
Ezhilarasi, P.N., Indrani, D., Jena, B.S., Anandharamakrishnan, C., 2013. Freeze drying
technique for microencapsulation of Garcinia fruit extract and its effect on bread
Leonard Mihaly Cozmuta: Conceptualization, Methodology, Inves quality. J. Food Eng. 117, 513–520.
tigation, Writing-Original draft preparation. Camelia Nicula: Writing - Haghighat-Kharazi, S., Kasaai, M.R., Milani, J.M., Khajeh, K., 2020. Optimization of
review &editing. Anca Peter: Supervision, Writing - review &editing. encapsulation of maltogenic amylase into a mixture of maltodextrin and beeswax
and its application in gluten-free bread. J. Texture Stud. 51 (4), 631–641.
Robert Apjok: Investigation. Agnieszka Jastrzębska: Methodology,
Han, M.R., Kwon, M.C., Lee, H.Y., Kim, J.C., Kim, J.D., Yoo, S.K., Sin, I.S., Kim, S.M.,
Writing - review &editing. Anca Mihaly Cozmuta: Investigation, 2007. pH-dependent release property of alginate beads containing calcium
Validation. carbonate particles. J. Microencapsul. 24 (8), 787–796.
Jackson, R.S., 2020. Fermentation. In: Jackson, R.S. (Ed.), Wine Science: Principles and
Applications. Academic Press, USA, pp. 461–572.
Declaration of competing interest Komlenić, D.K., Vedran, S., Jukić, M., 2012. Rheology. In: De Vicente, J. (Ed.), Influence
of Acidification on Dough Rheological Properties. InTech, UK, pp. 265–292.
The authors have no conflicts of interest to declare. The manuscript Larsen, N., Brøsted Werner, B., Vogensen, F.K., Jespersen, L., 2015. Effect of dissolved
oxygen on redox potential and milk acidification by lactic acid bacteria isolated from
has not been previously published not is it under consideration for a DL-starter culture. J. Dairy Sci. 98 (3), 1640–1651.
publication elsewhere. Lin, Y.-H., Wan-Shan, C., Duan, K.-J., 2010. Correlations between reduction–oxidation
potential profiles and growth patterns of Saccharomyces cerevisiae during very-high-
gravity fermentation. Process Biochem. 45, 765–770.
Acknowledgments Lippuner, A.D., Julou, T., Barral, Y., 2014. Budding yeast as a model organism to study
the effects of age. FEMS Microbiol. Rev. 38 (2), 300–325.
No outside funding was received for this work. The instrumental Liu, C.-G., Lin, Y.-H., Bai, F.-W., 2011. A kinetic growth model for Saccharomyces
cerevisiae grown under redox potential-controlled very-high-gravity environment.
support provided by the Scientific Research Center of Environment, Biochem. Eng. J. 56, 63–68.
Food and Health (CCESMAS) from Technical University of Cluj Napoca Liu, C.-G., Xue, C., Lin, Y.-H., Bai, F.-W., 2013. Redox potential control and applications
(Romania) and Food Microbiology Laboratory of S.C. SAN-V-TEST S.R.L. in microaerobic and anaerobic fermentations. Biotechnol. Adv. 31 (2), 257–265.
Mihaly Cozmuta, A., Jastrzębska, A., Apjok, R., Petrus, M., Mihaly Cozmuta, L., Peter, A.,
(Romania) is gratefully acknowledged.
Nicula, C., 2021. Immobilization of baker’s yeast in the alginate-based hydrogels to
impart sensorial characteristics to frozen dough bread. Food Biosci. 42, 101143.
Appendix A. Supplementary data Navrot, N., Holstborg, R.B., Hägglund, P., Povlsen, I.L., Svensson, B., 2018. New insights
into the potential of endogenous redox systems in wheat bread dough. Antioxidants
7 (12), 190.
Supplementary data to this article can be found online at https://doi. Noort, M.W.J., Bult, J.H.F., Stieger, M., 2012. Saltiness enhancement by taste contrast in
org/10.1016/j.jcs.2022.103516. bread prepared with encapsulated salt. J. Cereal. Sci. 55, 218–225.
Pasrija, D., Ezhilarasi, P.N., Indrani, D., Anandharamakrishnan, C., 2015.
Microencapsulation of green tea polyphenols and its effect on incorporated bread
References quality. LWT–Food Sci. Technol. 64, 289–296.
Smart, K.A., 2001. Biomarkers of yeast condition – predicting fitness to ferment. Institute
Alwazeer, D., 2020. Importance of consideration of oxidoreduction potential as a critical and Guild of Brewing Proceedings of the African Section Congress 8, 138–149.
quality parameter in food industries. Food Res. Int. 132, 109108. Stoycheva, T., Venkov, P., Tsvetkov, Ts, 2007. Mutagenic effect of freezing on
Bonjean, B., Guillaume, L.-D., 2003. 11 - Yeasts in bread and baking products. Yeasts in mitochondrial DNA of Saccharomyces cerevisiae. Cryobiology 54, 243–250.
food, first ed. Woodhead Publishing Limited, UK, pp. 289–307. Wei, C.J., Tanner, R.D., Malaney, G.W., 1982. Effect of sodium chloride on bakers’ yeast
Bryszewska, M.A., Tomás-Cobos, L., Gallegob, E., Paz Villalba, M., Riverab, D., Taneyo growing in gelatin. Appl. Environ. Microbiol. 43 (4), 757–763.
Saa, D.L., Gianotti, A., 2019. In vitro bioaccessibility and bioavailability of iron from Zhang, L., Chena, D.X., Boomb, R.M., Schutyser, M.A.I., 2018. Survival of encapsulated
breads fortified with microencapsulated iron. LWT–Food Sci. Technol. 99, 431–437. Lactobacillus plantarum during isothermal heating and bread baking. LWT–Food Sci.
Caldeo, V., McSweeney, P.L.H., 2012. Changes in oxidation-reduction potential during Technol. 93, 396–404.
the simulated manufacture of different cheese varieties. Int. Dairy J. 25, 16–20.
Capuani, A., Behr, J., Vogel, R.F., 2012. Influence of lactic acid bacteria on the
oxidation–reduction potential of buckwheat (Fagopyrum esculentum Moench)
sourdoughs. Eur. Food Res. Technol. 235, 1063–1069.
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