Journal of Cereal Science 107 (2022) 103516

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Journal of Cereal Science

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

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.

1. Introduction process to obtain better sensory characteristics. Lin et al. (2010)

The fermented food industry produces a wide range of products,
including baked goods, beverages, dairy, soy, fish, and coffee. As the
main stage in their production, fermentation is strongly influenced by
internal factors (i.e., the nature and amount of the ingredients) and
external factors (i.e., temperature, humidity, time). Fermentation
progress can be monitored by considering the evolution of a wide range
of parameters, such as redox potential, pH, titratable acidity, biomass
growth, specific volume, consumption of fermentable sugars, or CO2
production. Based on the operating diagrams, the fermentation process
can be optimized to obtain higher yields or develop more stable and
nutritious products with specific aroma, flavor, and color, or with
extended shelf-life (Alwazeer, 2020). The work of Caldeo and
McSweeney (2012) monitored the redox potential, temperature, and pH
during the simulated manufacture of different types of cheese over the
various steps of the cheese-making process. They found that monitoring
the redox potential allowed better control and standardization and
developed the aroma of the cheese. Later, Larsen et al. (2015) clarified
the relationship between the redox state in milk and the acidification
kinetics of lactic acid bacteria isolated from DL-starter cultures which is
important in the dairy industry for standardizing the milk acidification
explored the correlation between the reduction–oxidation potential
profiles and growth patterns of Saccharomyces cerevisiae during
high-gravity fermentation. As a result, the subtle transition of yeasts
from one growth phase to another was revealed. By studying the influ­
ence of lactic acid bacteria on the oxidation–reduction potential and the
proteolytic activity of buckwheat (Fagopyrum esculentum Moench)
sourdoughs, Capuani et al. (2012, 2013, 2014) were able to determine
the fermentation endpoint and the harvesting point, which is helpful in
the development of new cereal fermentation types as in gluten-free
sourdoughs. Moreover, their work indicated that the oxida­
tion–reduction profiles of different sourdoughs could be employed for
starter culture screening and cell density predictions. The interpretation
of the evolution profiles is very helpful to determine the time required
for batch feeding or ripening and the selection of the starter culture most
suitable for a specific sourdough. Various agents employed in reduction
(i.e. cysteine and ascorbic acid) or oxidation (L-ascorbic acid and lip­
oxygenase) are often used in bread-making to change the redox state of
the dough, aiming to influence the rheological properties of the dough
(Navrot et al., 2018).
A new trend in the bakery industry considers the encapsulation of
bioactive compounds to improve the baking process (Haghighat-Kharazi

* 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)

Formula 3 (reference - Mihaly Cozmuta et al., 2021)

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
bread (Bryszewska et al., 2019; Ezhilarasi et al., 2013; Pasrija et al.,
2015; Zhang et al., 2018). The capsules could influence the fermentation
stage by interfering with the physical, chemical, and microbiological
processes occurring in the dough. The encapsulated bioactive com­
pounds alter the redox balance of the dough and may result in bread
with poor sensorial characteristics due to their oxidizing (some metallic
ions such as Fe3+) or reducing (vitamins, carotenoids, flavonoids, and
Fe2+) potential (Alwazeer, 2020). An improved understanding of the
fermentation of such dough could help select the optimal parameters to
obtain bread with sensorial characteristics highly accepted by
consumers.
Our previous work (Mihaly Cozmuta et al., 2021) investigated the
immobilization of S. cerevisiae baker’s yeast cells in an alginate matrix in
order to protect them during the freezing – frozen storage – thawing
process and improve the sensorial characteristics of bread produced
from frozen dough. The 3-month frozen dough bread made with yeasts
immobilized in a calcium alginate matrix (CAYD) collected high, but not
maximum, average scores for crust color, firmness, taste, smell, and
overall acceptability.
In this context, this paper aims to identify and select the conditions
that improve the above-mentioned sensorial attributes. The paper is
organized as follows: (i) constructing operating diagrams based on the
evolution of the S. cerevisiae count, redox potential, pH, and specific
volume during fermentation, and the values associated with the highest
specific volumes of doughs were selected; (ii) baking loaves from the
investigated doughs with the selected parameters, which were subjected
to sensorial study; and (iii) determining the fermentation parameters
associated with the fresh and frozen dough bread that was the most
appreciated by the consumers.
2. Materials and methods
2.1. S. cerevisiae yeast cell immobilization
Beads made of dry yeast cells (DY) immobilized in an alginate matrix
with the characteristics described by Mihaly Cozmuta et al. (2021) were
used in the experiment. Briefly, the beads (CAY) were prepared from
alginate:yeast:water in a ratio of 2:5:100 (w:w:v) with an encapsulation
efficiency of 72.79% and carried a yeast cell load of 1.38 × 10− 7 CFU/g.
The equivalence between CAY beads and DY in terms of yeast cell load
was 4.70 g of CAY beads/1 g DY (dry matter basis).
2.2. Frozen dough preparation and storage
Three dough formulas were investigated, as depicted in Table 1. The
reference formula (formula 3) described the ingredients in the ratios that
led to the fresh and 3-month frozen dough loaves that received the
highest sensory scores, as described in our previous work (Mihaly Coz­
muta et al., 2021). The amount of yeast (Pakmaya, Romania) followed
the manufacturer’s recommendation, and the amount of beads added
was equivalent to the amount of yeast (4.70 g of CAY beads/1 g DY).
Another two dough types (formula 1 and formula 2) were prepared by
varying the ratios of salt (Salrom, Romania) or ascorbic acid (AsA,
Merck, Germany) (Table 1). Preliminary investigations were conducted
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
acteristics of the resulting bread.
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),
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,
to activate the yeast, then divided into smaller parts and kept for 6 h at
32 ◦ C and 75% relative humidity in a proofing chamber (Matina D 6040,
Bucharest, Romania).
The pH and redox potential of fresh and thawed dough pieces were
continuously monitored during fermentation. Simultaneously, periodic
sampling was conducted to determine the specific volume and yeast cell
count.
2.3. S. cerevisiae yeast cell counts
The viable yeast cells in the dough samples were counted according
to ISO 6887–4:2017. Samples of dough (5 g) were gently homogenized
in 45 mL of sterile buffered peptone water (0.1% w/w) in a stomacher,
and 0.1 mL from each serial dilution of suspension up to 10− 5 was
transferred onto Petri dishes with DRBC Agar (Dichloran Rose Bengal
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,
Germany) previously calibrated at two points in pH 7 and pH 4 buffer
solutions. The redox potential (related to the standard hydrogen elec­
trode, Eh) was measured with a redox electrode (HANNA Instruments,
pH/ORP/oC meter, HI991003, Italy). The pH and Eh electrodes were
punched directly into the dough sample and remained in place
throughout the experiment to avoid technical problems (signal errors,
air bubbles).
The specific volume of dough pieces during fermentation and the
final volume of the corresponding bread loaves were measured ac­
cording to the rapeseed displacement method (Mihaly Cozmuta et al.,
2015).
2.6. Bread baking and sensorial study
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

4
L. Mihaly Cozmuta et al.
thawed 810-g dough pieces were prepared according to Table 1, shaped
into a round form, proofed, and baked (details are presented in the
Supplementary Material). The bread loaves were marked with the codes
assigned to the dough pieces they originated from (Table 1) by replacing
the letter “D” (dough) with “B” (bread). The resulting bread loaves were
subjected to a sensorial study according to the protocols detailed in
Section 2.1. SM from Supplementary Material. The attributes of crust
color and firmness, crumb aspect, bread volume, smell, taste, and overall
acceptability were evaluated using a five-point hedonic scale (1 – dislike
extremely to 5 – like extremely). The overall sensory score of each
attribute was computed as the average of the individual scores provided
by the panelists.
2.7. Statistical analysis
All investigations were performed in triplicate, and the results were
expressed as mean ± standard deviation. One-way analysis of variance
(ANOVA) and Student’s t-test were used to assess the difference between
the two means. The probability value p ˂ 0.05 was considered statisti­
cally significant.
3. Results and discussions
Figs. 1 and 2 illustrate the influence of salt and ascorbic acid on the
growth of S. cerevisiae cells in fresh and frozen doughs, as well as the
redox potential, pH, and specific volume of the dough mentioned above
types. The standard deviations ranged between 0.04 and 0.19 for the
yeast cell count, 0.03–0.14 for the pH, 2.65–6.93 for the redox potential,
and 0.08–1.30 for the specific volume. Features of the yeast cells growth,
redox potential, pH and specific volume profiles in the investigated
dough types extracted from Figs. 1 and 2 are shown in Table 3SM, 4. SM,
5. SM and 6. SM from the Supplementary Material.
3.1. Biomass growth profiles
The growth profiles of S. cerevisiae cells revealed four distinctive
phases: lag, log, stationary, and decline (Figs. 1 and 2). In the lag phase,
the free yeast cells try to acclimate to the substrate, but some of them do
not adjust successfully, and the number of cells that have yet to begin to
divide is almost equal to the number that have died. Of all fresh doughs
made with free yeast cells, the shortest lag phase, around 20 min, was
observed in DYD-1.0 and DYD-3.0 (Table 3SM.). The brevity of their lag
phase indicated that the yeast cells contain the full complement of
fermentative enzymes (Jackson, 2020), and the switch from respiratory
to fermentative metabolism required only a short amount of time.
Conditions such as NaCl presence and low pH may disadvantage yeast
cells, as a result of the osmotic stress they experience (Wei et al., 1982;
Jackson, 2020). Although the NaCl and AsA are present simultaneously,
a rapid transition of yeast cells to the log phase can be observed for
DYD-1.0 and DYD-3.0, respectively. The small amounts in which they
were added seems to have a low negative impact on yeast cells, resulting
in short lag phase.
The elevated salt level in DYD-2.0 extended the lag phase to around
40 min, showing the difficulties of yeast adapting to the osmotic stress.
The accumulation of trehalose and glycerol is a part of the yeast’s
strategy to maintain the plasma membrane and stabilize the structural
proteins (Eardley and Timson, 2020; Jackson, 2020). The prolongation
of the lag phase was also observed for the doughs made with free yeast
cells subjected to the freezing – frozen storage – thawing process
(DYD-1.3, DYD-2.3, DYD-3.3). This effect is possibly due to the yeast
populations containing young and mature cells that were differently
affected by frozen storage. Old cells may handle stress more efficiently
than young ones (Lippuner et al., 2014), as the time required for re­
covery after thermal stress is longer for young cells, as part of their
energy is oriented toward repair mechanisms rather than growth (Smart,
2001). All these processes are directly correlated with the length of the
5
Journal of Cereal Science 107 (2022) 103516
lag phase. The fresh doughs made with encapsulated yeast cells
(CAYD-1.0, CAYD-2.0, CAYD-3.0) displayed longer lag phases compared
to the corresponding doughs made with free yeast cells. These length­
ened lag phases seem to result from the leakage rate of cells embedded in
the alginate beads into the dough rather than the yeast adjustment to the
substrate. Since the alginate is a pH-responsive polymer, it swells and
even dissolves in an environment containing anions with calcium af­
finity. The calcium ions from the alginate matrix leach out in an acidic
environment, leaving cavities that can be pathways to release the
embedded yeast cells (Han et al., 2007). The cracks and voids that
appeared on the surface of beads during the freezing – frozen storage –
thawing process (Mihaly Cozmuta et al., 2021) favored the diffusion of
yeast cells from beads into the dough and shortened the lag phase. An
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
the alginate beads and restricted the leakage of yeast cells, resulting in a
more extended lag phase of about 60 min.
After the yeast cells exit the lag phase, they undergo growth and
multiplication following a curve whose shape approximates an expo­
nential equation (log phase) at different rates, depending on the pre­
vailing conditions and the number of accumulated metabolites. After
they become active, the yeast cells quickly convert glucose and consume
oxygen through respiration to generate ATP for growth. Thus the pop­
ulation of viable cells is characterized by a low fermentation efficiency
(Liu et al., 2011). As oxygen depletion occurs, the metabolism of yeast
cells modulates from aerobic growth to anaerobic glucose fermentation
to ethanol and secondary metabolites, whose CO2 is essential in the
bread-making process. In the fresh doughs, the lowest growth rate of
8.48% occurred in the DYD-2.0 (Table 3SM) in response to the osmotic
stress induced by the high concentration of salt (1.2%), which caused
the plasmolysis of cells and retarded their multiplication. Bonjean and
Guillaume (2003) have also reported reduced fermentation at a salt
content above 1%. As expected, the highest multiplication rate was
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
growth (Echevarria et al., 2010) to a larger extent than in DYD-3.0
(24.94%), in which the amount of AsA is two times smaller. Interme­
diate values of 29.03%, 11.29%, and 29.57% were found for the growth
rates of yeast cells in the CAYD-1.0, CAYD-2.0, and CAYD-3.0 fresh
doughs, respectively, as the immobilization of the yeast constrained
their access to the substrate and therefore retarded growth. As seen in
Figs. 1 and 2, the doughs subjected to frozen storage for 3 months dis­
played higher yeast cell growth rates compared to the fresh doughs. This
can be accounted for by the lower density of yeast cells competing for
the substrate nutrients. In addition, in the case of the doughs made with
immobilized yeast, the corrosion of the bead walls that occurred during
the freezing – frozen storage – thawing process favored the faster release
of the yeast into the dough. The highest growth rate, 49.11%, occurred
in CAYD-1.3, the thawed dough that contained immobilized cells, while
in DYD-1.3, the corresponding dough made with free yeast, the growth
rate was 33.06%. The stress that occurred during the freezing – frozen
storage – thawing process affected the free yeast cells to a larger extent
than the immobilized ones, not only in terms of their viability but also
the quality of the surviving cells, given the reported mutagenic effect on
their mitochondrial DNA (Stoycheva et al., 2007). As outlined in
Table 3SM, a time range of 120–300 min is required to achieve the
highest growth rates of free and immobilized cells in fresh and thawed
doughs.
The lack of nutrients accompanied by the accumulation of toxic
metabolic by-products, such as mid-chain carboxylic acids (C8 and C10)
and their esters (Jackson, 2020), led to the stationary phase (Figs. 1 and
2), which is characterized by arrested growth, although the fermenta­
tion process still occurs. The cells maintain their viability without added
nutrients, depending on their physiological state. The yeast cells in
DYD-1.0, which were not stressed by high salinity, immobilization, or
the freezing – frozen storage – thawing process, extended their viability
L. Mihaly Cozmuta et al.
during the stationary phase by up to 80 min (Table 3SM). Shorter sta­
tionary phases of around 60 min are associated with the cells restrained
in the alginate matrix, subjected to freezing – frozen storage – thawing
process, or developed in acidic doughs. Similar to what was observed in
the growth phase, an increased salt level reduced the length of the sta­
tionary phase in DYD-2.0 to 40 min, while the combination between
salinity and freezing – 3-month frozen storage – thawing reduced it even
more, to 20 min in DYD-2.3. The protection of immobilized cells against
high salinity and freezing exerted by the alginate matrix is reflected by
stationary phases of around 40 min observed in CAYD-2.0 and
CAYD-2.3.
With the gradual accumulation of toxic metabolites (octanoic and
decanoic acids and their esters) and the reduction of nutrients, more
yeast cells die or become dormant than those that divide (Jackson,
2020), and the biomass enters the decline phase (Figs. 1 and 2). Three
patterns can be observed in the decline phase: (1) it was not achieved
during the investigated time range in DYD-1.0, DYD-1.3, CAYD-1.0, and
CAYD-3.3; (2) there was a smooth decline in DYD-1.3, DYD-3.3,
CAYD-3.0, and CAYD-1.3; and (3) an abrupt decline occurred in
DYD-2.0, DYD-2.3, CAYD-2.0, and CAYD-2.3.
3.2. Redox potential profiles
The redox profiles (Figs. 1 and 2) of the investigated doughs followed
either a bathtub curve (DYD-1.0, DYD-1.3, DYD-3.0, DYD-3.3), a
continuously declining curve (DYD-2.0, DYD-2.3, CAYD-2.0, CAYD-2.3),
or a declining curve followed by a plateau (CAYD-1.0, CAYD-3.0, CAYD-
1.3, CAYD-3.3). All initial redox potentials are above 100 mV
(Table 4SM). High values were associated with the lag phase, and log­
arithmic behavior at the onset of the lag phase was due to oxygen
incorporated into the dough during mixing. The oxygen triggered the
respiratory circuit and resulted in low fermentation efficiency (Liu et al.,
2011). The development of biomass in a high redox potential environ­
ment favors the synthesis of unsaturated fatty acids in cells that will be
used to build a well-structured and highly protective cell membrane (Liu
et al., 2011).
As the log phase developed, the electrons captured by the oxygen
dissolved in the dough could not compensate for the electrons released
via biomass formation (Lin et al., 2010), as the environment became
more reduced and the redox potential declined until it plateaued. In the
absence of oxidizing compounds in the dough, the redox potential could
drop to negative values. The work of Capuani et al. (2014) correlated the
negative slope of the dough redox profile with the initial yeast cell load,
as the low-inoculation dough showed a delay in the reduction step,
reduction rate, and minimal reduction value. In our study, the reduction
rate depended significantly on the initial cell load and stress factors that
could inhibit or delay their development. Even if they had almost the
same initial cell load of 4.01 × 105 CFU/g, DYD-1.0, DYD-2.0, and
DYD-3.0 displayed reduction rates of 0.78, 0.50, and 0.67 mV/min,
respectively (Table 4SM). The sharpest decline, observed in DYD-1.0,
can be attributed to a more favorable growth environment for the
yeast cells than in DYD-3.0 and DYD-2.0. In the corresponding frozen
doughs, lower reduction rates were calculated as 0.52 mV/min in
DYD-1.3, 0.29 mV/min in DYD-2.3, and 0.65 mV/min in DYD-3.3. The
reduction process was delayed in the doughs made with immobilized
yeast cells and reflected by the reduction rates, which were below those
made with free yeast cells. The reduction process occurs in CAYD-1.3 at
a higher rate (0.54 mV/min) than in the corresponding DYD-1.3 (0.52
mV/min), correlated with the growth rates (49.11% and 33.06%,
respectively) rather than with the initial yeast cell load (1.12 × 105
CFU/g and 1.21 × 105 CFU/g). A particular case is the doughs that
display a continuous decrease of the reduction potential (DYD-2.0,
DYD-2.3, CAYD-2.0, and CAYD-2.3). The life cycle of yeast expressed as
long lag phases, low growth and reduction rates, and short stationary
phases is probably responsible for the decrease.
The basin of the redox profile (i.e., the plateau of the curve) can be
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Journal of Cereal Science 107 (2022) 103516
associated with the stationary phase of the yeast growth curve.
Depending on the composition of the dough, it can continue with an
uprising branch or be maintained during the rest of the experiment.
The uprising branch of the redox profile can be associated with the
decline phase in the growth profile of yeast cells. Of all the redox pro­
files, this behavior is displayed only by DYD-1.0, DYD-1.3, DYD-3.0, and
DYD-3.3 (Fig. 1a, d, 1c, 1f), indicating slowed or stalled fermentation as
a result of yeast cell lysis and resulting accumulation of oxidative me­
tabolites in the dough (Liu et al., 2013). The concordance between the
redox profile and the yeast cell growth profile in DYD-1.0 and DYD-3.0 is
seen in the good alignment of the basins of the redox profiles and the
stationary phases in the growth profiles. The appearance of the uprising
branches in the doughs mentioned above can be correlated with severe
drops in the decline phases of the yeast cell growth curves (Figs. 1 and
2), which indicate the accumulation of large amounts of oxidative
compounds. The redox profiles of fresh and thawed doughs made with
immobilized yeasts not subjected to NaCl stress maintain their basins.
This can be explained by the absence (CAYD-1.0, CAYD-1.3, and
CAYD-3.0) or low values of the decrease rate of yeast cells (CAYD-3.3).
The doughs displayed different reduction capacities in terms of
minimal Eh values (Table 4SM). The redox potential in the fresh doughs
prepared with free yeast cells constantly decreased to negative values.
The lowest values, − 38 and − 37 mV, were recorded in DYD-3.0 and
DYD-2.0, compared to − 4 mV in DYD-1.0. In the corresponding thawed
doughs, the minimum Eh became less reduced, and higher minimal
values were reached: − 27, 62, and 75 mV. By comparison, the minimum
Eh in the fresh dough made with immobilized yeast cells (CAYD-1.0,
CAYD-3.0) had positive values. Furthermore, positive values dropped to
negative ones in the thawed doughs CAYD-1.3 and CAYD-3.3, indicating
an increase in the reduction power of the dough. This effect is related to
the highest growth rates in the study, which occurred in CAYD-1.3 and
CAYD-3.3 (Table 4SM). The behavior of the minimal Eh in CAYD-2.3
resembled that of free yeast doughs; its value (67 mV) was higher
than that in the fresh dough CAYD-2.0 (27 mV).
3.3. pH evolution profiles
Acidification fulfills important functions in bread in terms of the
modulation of dough rheological properties, flavor, and texture, passing
through a range of pH values close to the optimum for various enzymes
present in the dough that result in structural changes, retardation of
stalling, prevention of bread aging, and delay to bacterial spoilage
(Komlenić et al., 2012).
Fig. 1 shows continuously decreasing pH values of the fresh doughs
prepared with free yeast cells. The early pH curves show a small decline,
aligned to the lag phase of the corresponding yeast cells’ cells’ growth
profiles. Thus, in DYD-1.0, the biomass grew 0.2% in the first 20 min and
another 2.53% over the following 20 min, while the pH decreased by
0.45% and 2.40%, respectively. To a lesser extent, the same trend was
also observed in CAYD-2.0 and CAYD-3.0. Only a small part of the
fermentable sugars was fermented in the lag phase. The number of acidic
metabolites (such as succinic acid and acetic acid) released into the
dough was reduced, and the pH declined slightly. A good alignment can
be observed in DYD-1.0 and DYD-3.0 between the biomass growth, Eh,
and pH profiles: the exponential phases correspond to the rapid decrease
in Eh and pH, while the beginnings of the stationary phases were syn­
chronized with the Eh and pH basins. Regarding the thawed doughs with
free yeast cells, the pH declined at higher rates than in the counterpart
fresh doughs, while lower values of the minimum pH were achieved in a
shorter time (Table 5SM). This indicated more intense fermentation due
to the decrease of the yeast cell load and easier access to the substrate.
Doughs made with immobilized yeasts in an alginate matrix had
interesting pH profiles (Fig. 2) due to two parallel processes that
released products with opposite chemical properties. On the one hand,
the yeast cells on the surface of the beads detached easily, adjusted to the
dough, grew, and initiated the fermentation process with the release of
L. Mihaly Cozmuta et al. Journal of Cereal Science 107 (2022) 103516

acidic metabolites (acetic, lactic, propionic, butyric acids). Moreover, Table 2

the entrapped yeasts were released and contributed to the fermentation Parameters associated with the maximum specific volume of each dough type
process. On the other hand, the dissolution of the alginate-based beads extracted from operating diagrams (Figs. 1 and 2).
led to dough alkalization due to the presence of calcium ions. The AsA in Dough Maximum Yeast Eh, pH Proofing Retention of
CAYD-1.0, CAYD-1.3, CAYD-3.0, and CAYD-3.3 favored the quick type specific cells mV time the
dissolution of alginate-based beads, the doughs became more alkaline, volume, load, required to maximum
cm3/g *105 achieve the specific
and their initial pH rose. The switching of yeasts from respiration to early
CFU/g maximum volume,
fermentation explained the first plateau in the pH profiles, for which the specific minutes
acidic compounds released into the dough were responsible. As volume,
fermentation progressed, the dough became richer in acidic compounds, minutes
and the pH decreased. Fresh dough
The differences between the fresh and thawed doughs made ac­ DYD- 125.55 4.45 − 3 4.04 120 60
cording to formulas 1 and 3 resulted from the maximum pH values and 3.0
the time required to achieve and maintain them. In CAYD-1.0 and DYD- 110.23 4.86 7 3.85 180 60
CAYD-3.0, lower maximum pH values and shorter maintenance times 1.0
CAYD- 108.83 2.25 40 4.13 180 60
were expected, compared to those of CAYD-1.3 and CAYD-3.3, due to
3.0
the higher initial yeast cell load of CAYD-1.0 and CAYD-1.3 (Table 3SM), DYD- 97.19 4.35 − 16 4.18 300 20
which suggested more intense fermentation. Nevertheless, the experi­ 2.0
mental results turned out to be the opposite (Table 5SM). The freezing – CAYD- 90.11 2.18 27 3.93 180 60
frozen storage – thawing cycle reduced the strength of the hydrogen 1.0
CAYD- 67.31 2.04 41 3.96 200 60
bonds established between compounds in the yeast cell walls and the 2.0
alginate matrix (Mihaly Cozmuta et al., 2021) combined with higher
Thawed dough
multiplication rates in CAYD-1.3 and CAYD-3.3 (Table 3SM) account for
this inconsistency. For example, the maximum pH value of 3.96 was CAYD- 124.94 1.43 − 2 3.82 120 40
achieved in CAYD-1.3 after 40 min, corresponding to a pH increase of 1.3
DYD- 121.33 1.61 89 3.77 240 40
6.92% at a rate of 0.0065 min− 1, and maintained for about 20 min. In 1.3
CAYD-1.0, it took 60 min to reach the maximum pH value of 4.01, DYD- 121.33 1.44 − 18 3.83 240 40
corresponding to an increase of 6.48% at a rate of 0.0043 min− 1, and the 3.3
plateau was maintained for 40 min. A higher maximum pH value and CAYD- 120.18 1.33 − 2 3.91 180 40
3.3
wider plateau were also recorded in CAYD-3.3 compared to CAYD-3.0.
DYD- 116.16 0.88 72 4.11 180 40
The high salt content in CAYD-2.0 and CAYD-2.3 resulted in sluggish 2.3
or stalling dissolution of the alginate beads and yeast fermentative ca­ CAYD- 72.45 1.04 80 3.92 180 40
pacity and accounted for their declining pH profiles, in which the initial 2.3
pH is maintained for a certain time and followed by a constant and
smooth decline (Fig. 2b and e).
achieved after 6 h. Even the yeast population multiplied at a higher rate
in DYD-1.0 (34.42%) compared to DYD-3.0 (24.94%), which implies
3.4. Dough-specific volume profiles
more intense fermentation. A lower specific volume was reached in
DYD-1.0 (110.23 cm3/g), attributed to the difference between the
In our study, various dough formulations and storage types resulted
amounts of AsA in the dough formulas. When the amount of AsA is large
in different dough expansions (Figs. 1 and 2). Four distinct phases can be
(as in the case of DYD-1.0), the gluten network breaks during proofing
observed in all the specific volume profiles: slow increase, marked in­
under the pressure of CO2 and gas loss occurs. The 60-min retention time
crease up to a maximum value, plateau, and decline. The initial phases
for the maximum specific volume in DYD-1.0 is the same as in DYD-3.0.
of all the specific volume profiles were synchronized with the lag phase
The addition of around 1.2% salt (flour weight basis) in DYD-2.0
of the yeast cell growth patterns, while the maximum specific volumes
severely reduced the maximum specific volume of the dough to 97.19
were reached before the maximum growth rate of the yeast cells were
cm3/g and the retention time by one-third (20 min) because of the salt
achieved, with some exceptions. Thus, the maximum values of the
sensitivity of the yeast cells, improved dough cohesiveness, and elas­
specific volume and biomass growth were achieved in the same time in
ticity reduction (Chen et al., 2019). The greatest decrease in specific
DYD-2.0, DYD-1.3, and DYD-3.3, while in CAYD-2.3, a delay in the
volume occurred in DYD-2.0 (18.59%) followed by DYD-1.0 (17.38%)
development of specific volume was noticed. This discrepancy is related
and DYD-3.0 (16.99%). Lower yeast cell loads in the fresh doughs made
to the loss of gas from the dough. The yeast cells release CO2 during
with beads (CAYD-1.0, CAYD-2.0, and CAYD-3.0) were associated with
fermentation, the pressure from which expands the gluten network.
delays in bead leakage, slowing the multiplication and fermentation
Higher CO2 production does not necessarily result in an increased spe­
processes, and generating lower CO2 volumes and implicitly lower
cific volume, as retaining CO2 inside the dough requires balanced
specific volumes compared to the counterpart doughs containing free
gluten-network strength and elasticity. Factors such as salt, acidity, or
yeast cells (Table 6SM). The specific volume of CAYD-3.0, 108.83
high proteolytic activity depolymerize the gluten in molecules, weaken
g/cm3, seems to be the closest to the maximum volume of DYD-1.0,
the gluten strength, reduce its extensibility, and alter its ability to retain
110.23 g/cm3. The lowest specific volume (67.31 cm3/g) and expan­
the gas. In addition, freezing and frozen storage of dough reduce the
sion level (27.77%) were seen in the fresh dough CAYD-2.0, as the high
viability and fermentative capacity of yeast, while the reducing sub­
amount of salt in the composition slowed the leakage of yeast cells from
stance, glutathione, released by dead yeast cells weakens gluten strength
the beads. A longer, 60-min retention time of the maximum specific
and prevents CO2 retention (Collins and Haley, 1992).
volume occurred in CAYD-2.0 in response to the lower pressure exerted
The times required to achieve and retain the maximum specific
by the CO2 against the gluten network compared to the 20-min retention
volume are essential parameters in the bakery industry because they
time of the maximum specific volume in DYD-2.0. By comparison, the
influence the choice of the dough proofing time. Of all the fresh doughs,
reduction of the specific volumes and decrease rates in fresh doughs
DYD-3.0 had the highest specific volume, 125.55 cm3/g (Table 6SM),
(CAYD) is lower than their fresh counterparts (DYD).
reached after 2 h of fermentation, during which time the yeast popula­
Of all the frozen doughs, the highest specific volume of 124.94 cm3/g
tion multiplied by 10.97%. The maximum growth rate, 24.94%, was

7
L. Mihaly Cozmuta et al.
was achieved in CAYD-1.3, which is close to the highest value obtained
in DYD-3.0, 125.55 cm3/g. Although the yeast cell loads in DYD-1.3 and
DYD-3.3 (1.21 × 105 CFU/g and 1.05 × 105 CFU/g) are slightly higher
than those in CAYD-1.3 and CAYD-3.3 (1.12 × 105 CFU/g and 1.05 ×
105 CFU/g), higher specific volumes were achieved in the doughs made
with immobilized yeasts, CAYD-1.3 and CAYD-3.3. Shorter lag phases
(Table 3SM) combined with higher dough expansion rates in CAYD-1.3
and CAYD-3.3 suggest better cryoprotection of immobilized yeast cells,
which resulted in a more intense fermentation and higher values of
dough expansion than DYD-1.3 and DYD-3.3 (Table 6SM). The 20-min
reduction of the retention times of the maximum specific volumes and
higher declines and decline rates in the specific volumes occurred in the
case of frozen doughs as opposed to the fresh ones, as a response to a
higher CO2 pressure due to more intense fermentation and gluten
weakening during the freezing and thawing cycle.
3.5. Centralization of optimal parameters
Ranking of the dough formulas by taking into consideration the
values of the maximum specific volume resulted in the series (Table 2):
8
Journal of Cereal Science 107 (2022) 103516
Fig. 3. Appearance of the external and internal
structure of the bread loaves baked from fresh and 3-
months frozen dough pieces containing free and
immobilized yeast cells:
a) DYB-1.0, DYB-2.0, DYB-3.0 - bread baked from
fresh dough made with free yeasts cells; CAYB-1.0,
CAYB-2.0, CAYB-3.0 - bread baked from fresh
dough pieces made with immobilized yeasts cells;
b) DYB-1.3, DYB-2.3, DYB-3.3 - bread baked from 3-
months frozen dough pieces made with free yeasts
cells; CAYB-1.3, CAYB-2.3, CAYB-3.3 - bread baked
from 3-months frozen dough pieces made with
immobilized yeasts cells.
DYD-3.0 > CAYD-1.3 > DYD-1.3 = DYD-3.3 > CAYD-3.3 > DYD- 2.3 >
DYD-1.0 > CAYD-3.0 > DYD-2.0 > CAYD-1.0 > CAYD-2.3 > CAYD-2.0
Considering the proofing time required to achieve the maximum
specific volumes and their retention time, the following series resulted:
DYD-3.0 = CAYD-1.3 (120 min) < DYD-1.0 = CAYD-3.0 = CAYD-1.0 =
CAYD-3.3 = DYD-2.3 = CAYD-2.3 (180 min) < CAYD-2.0 (200 min) <
DYD-1.3 = DYD-3.3 (240 min) < DYD-2.0 (300 min)
DYD-3.0 = DYD-1.0 = CAYD-3.0 = CAYD-1.0 = CAYD-2.0 (60 min) >
CAYD-1.3 = DYD-1.3 = DYD-3.3 = CAYD-3.3 = DYD-2.3 = CAYD-2.3
(40 min) > DYD-2.0 (20 min)
Several aspects of the parameters associated with the maximum
specific volumes of the investigated doughs are discussed in the section
3.5. SM from the Supplementary Materials.
3.6. Bread sensorial study
The baked bread loaves are presented in Fig. 3, and the results of the
L. Mihaly Cozmuta et al.
sensorial study are depicted in Fig. 4. The sensorial study is detailed in
the section 3.6. SM from Supplementary Material, aspects related to the
final volumes of bread loaves being discussed in this section. Different
trends were observed when the final volumes of bread loaves were
compared to the highest specific volumes of the doughs from which they
originated. The following series resulted from the average scores given
by the panelists to the volume of the fresh bread loaves (Fig. 4a):
DYB-3.0 > DYB-1.0 > CAYB-3.0 > DYB-2.0 = CAYB-1.0 > CAYB-2.0,
Which largely overlaps the series of maximum specific volumes of
the corresponding doughs (Table 2).
DYD-3.0 > DYD-1.0 > CAYD-3.0 > DYD-2.0 > CAYD-1.0 > CAYD-2.0
The bread loaves baked from frozen doughs were ranked in terms of
volume as (Fig. 4b):
CAYB-1.3 > CAYB-3.3 > DYB-3.3 > DYB-1.3 > CAYB-2.3 > DYB-2.3
While the series of maximum specific volumes of corresponding
doughs (Table 6 SM) was:
CAYD-1.3 > DYD-1.3 = DYD-3.3 > CAYD-3.3 > DYD- 2.3 > CAYD-2.3
A possible explanation for the discrepancy is the protective effect of
the alginate walls on the yeast cells during baking, which resulted in
9
Journal of Cereal Science 107 (2022) 103516
Fig. 4. Hedonic sensory evaluation of fresh and 3-
months frozen-dough bread made with free and
immobilized yeast cells:
a) DYB-1.0, DYB-2.0, DYB-3.0 - bread baked from
fresh dough made with free yeasts cells; CAYB-1.0,
CAYB-2.0, CAYB-3.0 - bread baked from fresh
dough made pieces with immobilized yeasts cells;
b) DYB-1.3, DYB-2.3, DYB-3.3 - bread baked from 3-
months frozen dough pieces made with free yeasts
cells; CAYB-1.3, CAYB-2.3, CAYB-3.3 - bread baked
from 3-months frozen dough pieces made with
immobilized yeasts cells.
prolonged fermentative activity, enhanced gas production, and conse­
quently, in the larger volumes of CAYB-1.3 and CAYB-3.3.
Comparing the average scores given to the CAYB-1.3 bread in the
present study (Fig. 4b) and the CAYB-3.3 bread in our previous study
(Mihaly Cozmuta et al., 2021) indicated that doubling the amount of
AsA in the dough formula resulted in improved scores for crust color,
crumb texture, volume, smell, and overall acceptability, while crust
firmness and taste received the same high scores.
4. Conclusions
Distinctive trends in biomass growth, redox potential, pH, and spe­
cific volume profiles were observed depending on bread dough formula
and storage. Among all the fresh doughs, the highest specific volume
was displayed by the reference dough made with free yeast cells (DYD-
3.0) at a negative redox potential (− 3 mV) with a pH around 4.04,
following 2 h of proofing. The highest specific volume of fresh dough
made with immobilized yeast cells (CAYD-3.0), characterized by a lower
yeast cell load, required the combination of high positive redox potential
(40 mV), a slightly higher pH (4.13), and a longer proofing time (3 h).
Immobilized yeast cells in alginate are recommended to prepare 3-
month frozen dough accompanied by twice the amount of ascorbic
acid (CAYD-1.3), which favored yeast cell leaching during proofing. In
this case, the highest specific volume of the doughs involved a negative
L. Mihaly Cozmuta et al.
redox potential (− 2 mV), a more acidic pH (3.82), and a shorter proofing
time (2 h).
In terms of sensory attributes, doubling the amount of salt above the
reference value negatively affected both fresh and frozen doughs. The
saline stress altered the viability of the yeast cells, disrupted the gluten
network, and resulted in bread with poor sensory characteristics. In
comparison, doubling the amount of ascorbic acid above the reference
value particularly favored the frozen dough made with immobilized
cells (CAYD-1.3), resulting in bread loaves with sensorial characteristics
that scored higher than those made with free yeast cells and were highly
accepted by the consumers.
CRediT author statement
Leonard Mihaly Cozmuta: Conceptualization, Methodology, Inves­
tigation, Writing-Original draft preparation. Camelia Nicula: Writing -
review &editing. Anca Peter: Supervision, Writing - review &editing.
Robert Apjok: Investigation. Agnieszka Jastrzębska: Methodology,
Writing - review &editing. Anca Mihaly Cozmuta: Investigation,
Validation.
Declaration of competing interest
The authors have no conflicts of interest to declare. The manuscript
has not been previously published not is it under consideration for
publication elsewhere.
Acknowledgments
No outside funding was received for this work. The instrumental
support provided by the Scientific Research Center of Environment,
Food and Health (CCESMAS) from Technical University of Cluj Napoca
(Romania) and Food Microbiology Laboratory of S.C. SAN-V-TEST S.R.L.
(Romania) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jcs.2022.103516.
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