foods

Article
Impact of Storing Condition on Staling and Microbial Spoilage
Behavior of Bread and Their Contribution to Prevent Food
Waste
Thekla Alpers 1 , Roland Kerpes 2, *, Mariana Frioli 2 , Arndt Nobis 3 , Ka Ian Hoi 3 , Axel Bach 4 , Mario Jekle 1
and Thomas Becker 1,2,3

1 Research Group Cereal Technology and Process Engineering, Brewing and Beverage Technology,
Technical University of Munich, 85354 Freising, Germany; [email protected] (T.A.); [email protected] (M.J.);
[email protected] (T.B.)
2 Research Group Beverage Biotechnology, Brewing and Beverage Technology, Technical University of Munich,
85354 Freising, Germany; [email protected]
3 Research Group Raw Material Based Brewing and Beverage Technology, Brewing and Beverage Technology,
Technical University of Munich, 85354 Freising, Germany; [email protected] (A.N.);
[email protected] (K.I.H.)
4 Science Journalist, 50670 Cologne, Germany; [email protected]
* Correspondence: [email protected]; Tel.: +49-8161-71-3277

Abstract: The high loss rate of bread is generally known to contribute to the alarmingly high
numbers in worldwide food waste. Correct storage techniques are believed to enable the reduction
of preventable food waste. Therefore, the influence of storage parameters on staling and spoilage
behavior of German bread within the limits of common household methods was investigated in
this study. The aim was to generate reliable data for staling and spoilage using different storage
methods (PE-layered microperforated paper bag, plastic bag, and fridge and bread box) to bridge the





 gap between consumer’s needs and scientific research questions. Everyday routines of life, such as
Citation: Alpers, T.; Kerpes, R.; Frioli, visual inspection, were compared with microbiological techniques and were found to represent an
M.; Nobis, A.; Hoi, K.I.; Bach, A.; adequate tool for microbial safety control. Visually undetectable fungal growth has not been found
Jekle, M.; Becker, T. Impact of Storing to result in the production of mycotoxins (fumonisins B1 and B2 and ochratoxin A) in quantifiable
Condition on Staling and Microbial or harmful concentrations. Thus, disgust should prevent any foodborne health risks as the visual
Spoilage Behavior of Bread and Their appearance should lead to avoiding the consumption of spoiled food before mycotoxins are produced
Contribution to Prevent Food Waste. in amounts causing adverse health effects within the limits of this experimental setup. Additionally,
Foods 2021, 10, 76. https://doi.org/ the storage temperature especially was found to influence the kinetics of staling processes, as a
10.3390/foods10010076
reduction accelerated the staling process. Further, crumb moisture loss was found to contradict a long
shelf life but, on the other hand, an elevated humidity was shown to provoke excessive microbial
Received: 2 December 2020
growth and should therefore be observed when designing suitable storage methods. Further, the
Accepted: 29 December 2020
correct choice of the bread type stored and a good sanitary practice represent simply accessible ways
Published: 2 January 2021
to prolong the storage period of bread loaves.
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai- Keywords: shelf life; household storage methods; firming; texture analysis; mycotoxins; sourdough;
ms in published maps and institutio- wheat pan bread; mixed-type sourdough bread; food waste
nal affiliations.

1. Introduction
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
Staling and microbial spoilage of bread are the two main reasons for the alarmingly
This article is an open access article
high contribution of bakery products to food waste all over the world (34.7% of the total
distributed under the terms and con-
amount of baked goods in Germany in 2015) [1]. Within the storage period of bread,
ditions of the Creative Commons At- several important changes contribute to the decreasing consumer acceptance of stored
tribution (CC BY) license (https:// bread. These changes include physicochemical processes such as the crumb firming
creativecommons.org/licenses/by/ process, water migration within the crumb, crust and the environment, the loss of flavor
4.0/). and microbial spoilage. Even though there has been respectable research addressed to this

Foods 2021, 10, 76. https://doi.org/10.3390/foods10010076 https://www.mdpi.com/journal/foods

Foods 2021, 10, 76 2 of 15

topic over the years, there is a lack of published research on the optimal consumer storage
conditions for bakery products to minimize waste.
In general, the staling process describes the mechanism of bread aging and is initiated
immediately after baking. With the termination of the input of thermal energy, phase
transition processes occur and modify the texture of bread. During the first hours after
baking, the recrystallization of amylose positively impacts the solidification of the crumb
structure, whereas amylopectin, the second main macromolecule accounting for the starch
fraction in flour, crystalizes over a longer period of days [2–4]. This retrogradation process is
also responsible for the redistribution of water on the molecular level as the recrystallization
of starch is accompanied by the increased formation of B-type crystalline polymorphs,
which are capable of immobilizing more water molecules than A-type crystals [5]. Thus,
water is removed from the gluten network, leading to a less elastic and firmer texture [6].
Further water migration processes occur on a macrostructural level as water is exchanged
within the moisture crumb and the dry crust according to the differences in vapor pressures
between crumb and crust until the mass equilibrium is reached [7]. The long-term processes
of starch retrogradation and water migration are further accompanied by the evaporation
of volatile components, leading to a change in consumer perception even within the first
days of storage [8]. The physicochemical changes taking place during the storage period
of bread can be traced by textural analysis. Transformations can be quantified by human
and instrumental analysis, including sensory evaluations, uniaxial compression testing
(according to AACCi method 74-10A) or dynamic mechanical analysis.
Beside textural transformations, consumer acceptance is affected by microbial spoilage.
As bread represents an excellent source for microbiological growth, it is likely to be sub-
jected to fungal contamination, limiting the shelf life. The intrinsic composition of bread
(≈40%(w/w) water content, pH = 5.5–6.0 and an aw -value within 0.94–0.97) facilitates
fungal growth [9]. As spores are commonly considered to be inactivated during the baking
process [10], contamination generally arises from the surrounding air, machines, workers
(production and sale), the consumer or the storage environment [9,11,12]. Such contami-
nations lead to fungal growth, which can result in undesired changes causing consumer
rejection due to the development of mycelium or the accumulation of mycotoxins in the
bread matrix, which can cause adverse health outcomes involving carcinogenic or geneti-
cally harmful effects [13]. Commonly detected fungi, molds and bacteria include Penicillium
spp., Aspergillus spp., Bacillus spp. and Cladosporium sp., with spoilage having been shown
to be dominated by Aspergillus spp. in warmer countries, whereas Penicillium spp. predom-
inate under moderate climate conditions [9]. A review of common bread spoilage fungi
is available [13]. In order to avoid preventable food waste, several preservation methods
are commonly involved in production, handling and storage processes. Beside adequate
and sanitary production conditions, the usage of sourdough has been reported to reduce
the growth rate of fungi, thus prolonging the shelf life of bakery products [14–17]. Such
procedures could help reducing the alarmingly large amount of worldwide food waste,
together accounting for one third of the food produced for human consumption. Bread
and cereal products contribute to food waste with a loss rate of 26.3% during production,
retail and wholesale and consumption in Germany [18]. Further, storage conditions can
significantly affect the staling and spoiling behavior of bread. Parameters such as storage
temperature and humidity are well-known to influence the retrogradation process and
growth rate of microorganisms [19–21].
The aim of this study is therefore to evaluate the impact of different storage parameters
on the shelf life of bread. It is hypothesized that a targeted selection of storage parameters
within the limits of ordinary household storage methods can attribute to a prolonged
shelf life for commonly consumed German bakery products, such as pan wheat bread and
mixed-type sourdough bread. Therefore, the impact of four different storage methods was
compared in terms of firming rate, changes in the visual appearance, the concentration of
fungi measured by plate counting and the accumulation of mycotoxins during a storage
Foods 2021, 10, 76 3 of 15

period of ten days. This could help solving uncertainties among consumers regarding
microbial safety and reduction of food waste due to undesirable textural appearance.

2. Materials and Methods

2.1. Bread Storage and Sampling
In order to represent everyday life circumstances, pan wheat bread and mixed-type
sourdough bread were purchased at a local bakery store in Freising, Germany. All sam-
ples originated from one manufacturing batch. According to the supplier, the wheat
bread contained wheat flour, water, sodium chloride and yeast, whereas the mixed-type
sourdough bread contained rye and wheat flour (70:30, w/w), water, sourdough, sodium
chloride and S. cerevisiae sp. After purchasing, four different common storage methods were
compared: (1) metal bread box (BB) at 22.0 ± 0.5 ◦ C, (2) polyethylene (PE)-layered microp-
erforated paper bag with fine ventilation holes every 1.5 cm (purchased at a local bakery) at
21.3 ± 0.4 ◦ C (LP), (3) low-density polyethylene (LDPE)-plastic bag (250 × 350 × 0.09 mm,
dm-folien GmbH, Reutlingen, Germany) at 21.9 ± 0.5 ◦ C (PR) and (4) LDPE-plastic bag
at 8.2 ± 0.2 ◦ C, representing storage in a fridge (PF). The temperature and moisture for
the storage method applied were permanently logged using a temperature and humidity
logger (OM-EL-USB-2-LCD, Omega, Deckenpfronn, Germany). An exemplary storage
setup can be seen in Figure A1. In order to provide enough sample material, two bread
loaves were stored together in one container for the pan wheat bread, whereas one loaf of
bread was stored per container for the mixed-type sourdough bread. For a period of ten
days, bread slices of 1.25 ± 0.25 cm were obtained daily from the open end of the breads,
imitating typical consumer behavior. The sampling and handling of slices was performed
with great care, using gloves and deploying surface disinfection methods after taking each
sample, to avoid any contamination from external sources and cross-contamination.

2.2. Initial Bread Characterization

An initial characterization of the purchased breads was conducted on day 0 of the
storage period. The pH value and total titratable acidity (TTA) were analyzed accord-
ing to the guidelines of the International Association for Cereal Science and Technology
(ICC) [22], using a pH meter (206-pH2, Testo SE & Co. KGaA, Lenzkirch, Germany) and
a Titrator (Titroline 6000, SI Analytics GmbH, Mainz, Germany) equipped with 0.1 M
NaOH (Merck KGaA, Darmstadt, Germany). Water activity (aw -value) was measured
with an electronic hygrometer (Aqua Lab, Decagon Devices, Pulmann, WA, USA). Using
the chilled-mirror dew-point technique, the aw -value was determined and previously
calibrated with standard solutions of NaCl (6 M, VWR International GmbH, Darmstadt,
Germany) and KCl (0.5 M, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) of known
activity. All measurements were performed at a sample temperature of 25 ◦ C.

2.3. Moisture Determination

The moisture content of the sampled slices was determined using an oven drying
method at 130 ◦ C for 16 h. The moisture content was calculated as the weight difference
in relation to the original sample weight. For standardization of the sample prepara-
tion, the samples were stored in sealed plastic bags overnight at −33 ◦ C before moisture
measurements were conducted.

2.4. Texture Profile Analyses

Crumb firming behavior was accessed using instrumental texture profile analysis.
Two bread slices (wheat bread: first slice of two breads which were stored under the same
conditions; mixed-type sourdough bread: halves of the first slice stacked upon each other),
each with a thickness of 12.5 mm, were analyzed using a Texture Analyser (TVT-300 XP,
5 kg load cell, Perten Instruments, Hägersted, Schweden). The firmness of the samples was
measured in two compression cycles to up to 40% of the original height. The following
test settings were applied: a test speed of 1.7 mm/s, a post-test speed of 1.0 mm/s, a
Foods 2021, 10, 76 4 of 15

trigger force of 0.05 N and a relaxation time of 5 s between the measurement cycles. The
measurements were performed after sufficient resting time in order to compensate potential
temperature differences.

2.5. Plating and Identification of Spoilage Organisms

The count of viable and cultivable yeast was evaluated following the ICC standard
method No. 146 for yeasts. The sampled slices were prepared for microbiological analysis
by grinding with liquid nitrogen to allow for a homogeneous and complete extraction of
yeasts and molds during the consecutive extraction process. The extraction was performed
with Ringer’s solution (VWR International GmbH, Darmstadt, Germany). Suitable dilu-
tions of 1 mL (10−2 and 10−4 ) were spread on malt extract agar and incubated at 28 ◦ C
for 2 days. The progress of microorganism growth was documented and the individual
colonies were identified microscopically. Isolation of individual colonies was performed
for further identification.

2.6. Mycotoxins
2.6.1. Fumonisins B1 and B2
Analysis for the fumonisins B1 and B2 was performed by liquid chromatography
with tandem mass spectrometry (LC-MS/MS) according to the method developed by [23].
First, 0.5 g of the grinded samples was extracted using 2 mL of extraction solution (acetoni-
trile/water/acetic acid 79:20:1, v/v/v). After 90 min of extraction using a REAX2 shaker
(Heidolph Instruments GmbH & CO. KG, Schwabach, Germany), the solid parts were
removed by centrifugation (2 min at 3000 rpm, Eppendorf AG, Hamburg, Germany) and
the extracts were diluted 1:1 (v/v) with a dilution solvent (acetonitrile/water/acetic acid
20:79:1, v/v/v). An injection volume of 5 µL was filtered using a 0.45 µm membrane and
injected into the LC-MS/MS system operating at a flow rate of 1 mL/min. The system used
for detection and quantification included a 1200 series HPLC-System (Agilent, Waldbronn,
Germany) consisting of a HiP-ALS SL autosampler, a 1200 series bin pump module, a
1200 series degasser and a 1100 series column oven, coupled to the Triple Quad 4500 MS
(SCIEX, Darmstadt, Germany). The ion spray voltage was set to 4.000 V, the curtain gas
pressure was set to 10 psi, the nebulizer gas pressure was 50 psi and the heater gas pressure
was 50 psi. The turbogas temperature was set to 550 ◦ C. The measurement was performed
in the multiple reaction monitoring (MRM) mode in positive mode. Further, the system
was equipped with a Gemini® C18 column, 150 × 4.6-mm i.d., 5-µm particle size column
(Phenomenex, Torrance, CA, USA). Eluent A was a 5 mM ammonium acetate, composed of
methanol/water/acetic acid 10:89:1, v/v/v) and eluent B was a 5 mM ammonium acetate,
composed of methanol/water/acetic acid 97:2:1, v/v/v). The bivariate gradient was set
as follows: 2 min at 100% eluent A followed by a linear increase of eluent B to 100% over
12 min, followed by an isocratic phase of 3 min at 100% eluent B and finally a 4 min
re-equilibration phase at 100% eluent A. Isotope-labeled internal standards of fumonisins
B1 and B2 were used for the quantification. The limits of detection were 11.2 µg/kg
and 8.2 µg/kg for fumonisins B1 and B2 respectively. The limits of quantification were
30.3 µg/kg and 22.7 µg/kg, following the abovementioned order.

2.6.2. Ochratoxin A (ELISA)

For determination of ochratoxin A, RIDASCREEN® Ochratoxin A 30/15 (R-Biopharm
AG, Darmstadt, Germany) was conducted as a competitive ELISA. The method was carried
out according to the manufacturer’s guidelines [24].

2.7. Statistical Analysis

All measurements were performed in triplicates. The stated standard deviation
accounts for the deviation between triplicates. Statistical evaluation was performed using
Origin (2018b, OriginLab Corporation, Northampton, MA, USA).
Foods 2021, 10, 76 5 of 15

3. Results and Discussion

3.1. Initial Bread Characteristics
Since the aim of this study was to elaborate the spoilage and staling behavior of bread
under everyday life circumstances, the bread analyzed in this study was purchased in
a local bakery. An initial characterization was performed to access the main parameters
known to impact the storage behavior of bread. For the analyses, a representative initial
sample was taken of each bread type on day 0. The results of the initial specification are
presented in Table 1 in terms of moisture content, water activity (aw -value) and acidity (pH-
value and TTA). These intrinsic factors are well known to be decisive for microbial growth.
The aim of the initial characterization was to predict the likelihood of spoilage and staling
for both bread types. It appears that the moisture content of the mixed-type sourdough
bread (48.59% ± 0.01%) was slightly higher compared to the water content of the pan wheat
bread (45.38% ± 2.60%). These differences were likely caused by the different flour types
and recipes used for the bread production. In general, the higher content of non-starch
polysaccharides (pentosans, β-glucan, lignin and cellulose, arabinoxylan) in rye flour leads
to higher water-absorbing properties [25] and thus to a higher water addition. Further, a
better gelling potential has been reported for rye-originated arabinoxylans compared to
wheat endosperm-originated arabinoxylans due to structural differences [26,27]. Therefore,
the higher moisture content of the mixed-type sourdough bread in this experiment was
likely to be caused by the higher water addition. Regarding the influence of the different
moisture contents on the storage behavior, higher water content is known to affect the
staling process but does not determine the spoilage process. For the latter, water activity is
the decisive factor. The water activity is defined as the ratio of the vapor pressure of water
over a substrate related to that over pure water at the same temperature and pressure. A
high aw -value can promote microbial growth, whereas high osmotic pressure can result
in dehydration, leading to a lack of growth ability and potential cell death. Thus, water
activity is a limiting factor for food spoilage. Having a rather high water activity, bread is
subjected to all kinds of spoilage since the lower limits for bacteria, yeast and molds are
comparatively low. The aw -values of both bread types are presented in Table 1 and suggest
that both bread types would be highly exposed to food spoilage [9]. According to hurdle
technology, which is commonly used to evaluate the microbial safety of food and is based
on the combination of several preservation factors, the acidity can be used to prolong the
shelf life of food products and counteract a high spoilage possibility due to high moisture
content [28]. From Table 1 it can be seen that the pH value of the mixed-type sourdough
bread was lower compared to the pan wheat bread and the total titratable acidity was
comparably high. Both values were within the advised range of typical values for the
respective product type [29]. In general, the higher acidity of the mixed-type sourdough
bread has its origin in the incorporation of sourdough. During the sourdough fermentation
step acids are produced which contribute to the taste and a longer shelf life [17,30]. The
use of sourdough is necessary to produce rye bread, as this is a common way to reduce
the enzymatic activity of α-amylase [31]. The higher acidity of the mixed-type sourdough
bread might have contributed to a lower spoilage probability and a better microbial stability
in the storage trials performed within this study.

Table 1. Results of initial bread characterization (x ± STD, n = 3).

Total Titratable Acidity

Moisture Content (%) aw -Value ( ) pH-Value ( )
(mL 0.1 M NaOH)
Pan wheat bread 45.38 ± 2.60 a 0.98 ± 0.01 a 5.48 ± 0.03 a 4.18 ± 0.31 a
Mixed-type sourdough bread 48.59 ± 0.01 a 0.97 ± 0.01 b 4.48 ± 0.06 b 9.11 ± 0.28 b
a,bMean values ± STD labelled with a different letter in the same column are significantly different according to Tuckey’s test on a
significance level of α = 0.05.
Foods 2021, 10, 76 6 of 15

3.2. Microbial Stability of Breads under Different Storage Conditions

To access the microbial stability of bread under different storage conditions, two
different methods were applied in this study. Visual inspection of the bread loaves was
performed and validated using microbiological techniques. In this setup, the visual appear-
ance was meant to represent the consumer’s test possibilities and the results were related to
the results of plate counting experiments as a common reference technique for the identifi-
cation of microbial spoilage. By comparing the spoiling results of the consumer’s technique
and those of the laboratory approach, the question of whether visual appearance represents
a suitable tool for the consumer to judge microbial safety was able to be answered.
Initial plate counting experiments confirmed the microbial safety at the beginning
of the storage period. As expected, no yeasts were present on day 0. During the whole
storage period of ten days, the mixed-type sourdough bread was shown to be more stable
compared to the pan wheat bread. Spoilage of mixed-type sourdough bread occurred
significantly later than for wheat pan bread and in only one of the four different storage
methods. For mixed-type sourdough bread, no growth of molds was visually detectable
until day 10 for any kind of storage method (see Figure A2). Only the storage in plastic
bags resulted in spoilage after day 10 (n = 1) or 13 (n = 3). Using microbiological analysis,
the spoiling organisms were identified as Penicillium spp. As the main purpose of this study
was to test different ordinary household storage methods for their suitability for prolonging
the shelf life of bread, the following comparison focuses on the spoilage behavior of pan
wheat bread. For this bread type, substantial differences were observed between the
different storage methods. Storage in a plastic bag at room temperature was the first
storage method subjected to visually detectable mold growth (see Figure A3). The visual
evaluation revealed the growth of molds from day 4 (n = 1) and 5 (n = 3) on. The molds
were found to grow on the wheat bread crust, while the crumb was without any mycelium
until day 10. This is believed to be caused by the experimental setup, as the slice subjected
to the surrounding atmosphere was renewed after every 24 h. The plate counting revealed
the presence of molds even before their visual appearance. Penicillium spp. showed
exponential growth after a long lag phase from day 4 (5 colony forming units (CFU)) on
and Cladosporium spp. was detected at day 8. In general, Penicillium spp. are known to
commonly dominate the microbial spoilage on bread under moderate climate conditions [9].
The excessively humid atmosphere (89.9% rh) dramatically provoked the growth of molds.
Even the sourdough containing mixed-type bread was subjected to Penicillium spp. after
10 days of storage in a plastic bag at room temperature. No other storage method caused
the spoilage of mixed-type sourdough bread.
The storage in bread boxes caused a longer lag phase and slower growth rates. The
first visual appearance of mycelium appeared at day 4 (n = 1), 5 (n = 2) or 6 (n = 3). Bread
boxes might commonly be expected to represent the most stable storage method, but
within this experiment they were shown to provoke a highly humid atmosphere (89.0% rh).
Even waterlogging was found to occur due to the limited air circulation underneath the
bread loaves, promoting the growth of molds at this position. Consequently, molds were
primarily found on the bottom of the breads, where moldy build-up of damp was found to
be strongest. Using plate counting experiments, Aspergillus spp. were detected for the first
time at day 2 (3 CFU) and presented exponential growth until day 10. As Aspergillus spp.
are unlikely to dominate the spoilage under moderate conditions, but are ubiquitously
present in the form of endospores, it is likely that the Aspergillus spp. infection occurred as
a secondary infection. This organism would probably have infected the plastic bag-stored
wheat bread as well, but the Penicillium spp. dominated the mixed culture there. Beside
Aspergillus spp., Penicillium spp. were identified on the wheat pan bread stored in the bread
boxes and were found to grow exponentially from day 6 on. Further, Cladosporium spp.
were detected at day 8.
For paper bag storage, the formation of a mycelium was detectable from day 5 (n = 2)
or 6 (n = 3) on. In comparison with the other storage methods, the humidity inside the
paper bag was found to be significantly lower due to the higher water permeability of
Foods 2021, 10, 76 7 of 15

the PE-layered microperforated paper (80.5% rh). This was found to reduce the growth
rate of Penicillium spp., leading to the first appearance of Penicillium spp. for the plating
experiments on day 6. Aspergillus spp. were detected for the first time on day 1 (n = 1,
1 CFU) or 2 (n = 3, 2 CFU) and presented exponential growth until day 10, probably due to
the same reasons as for the appearance of Aspergillus spp. in the bread box.
It is generally known that the growth of molds is preventable with storage at re-
frigerator temperature. Therefore, the storage of bread in plastic bags under refrigerator
conditions was included in the comparison of storage methods as well. Even though the
plastic bag was shown to promote the strongest spoilage at room temperatures, no mold
growth was detected upon storage in the same wrapping at 8 ◦ C for a storage period of
10 days by visual inspection nor plate counting. In this case, temperature was shown to be
the limiting factor, as the measured humidity was comparable to the humidity measured
in the plastic bags stored at room temperature.
Further experiments were performed to evaluate the effect of sanitary conditions
on bread spoilage. A moldy slice of wheat bread and a non-moldy slice of mixed-type
sourdough bread were placed together in a plastic bag and stored at room temperature.
The moldy wheat pan bread slice was used to simulate a high mold load in the storage
container as would be the case for an insanitary storage environment. The successful
cross-contamination of mixed-type sourdough bread after 4 (n = 1) to 5 days (n = 3) proved
the strong impact of the hygienic conditions during bread storage (see Figure A2).
From the results it is apparent that humidity and temperature varying in the range
of typical household storage methods are the main factors influencing the microbial shelf
life of bread. Reducing the temperature to refrigerated temperatures was shown to be able
to decelerate the growth rate of fungi. Further, the occurrence of high humidity due to
inappropriate wrapping materials or container design was found to promote microbial
spoilage. Consequently, both factors should be considered when designing suitable storage
methods for bread. Further, a good sanitizing practice was shown to be able to prolong
the shelf life of bread, as a high load of molds was shown to reduce the storage time
significantly. Visual testing was found to be less sensitive for the detection of microbial
growth. In general, the presence of molds and yeasts causes disgust, though they do
not represent harm for human health in general. Nevertheless, food safety is affected
by mycotoxins, the production rate of which is not linked to the same parameters as
fungal growth. Therefore, food safety can only be judged by testing for the occurrence of
mycotoxins.

3.3. Relation between Microbial Growth and Mycotoxin Production

The most relevant mycotoxins in food are aflatoxins, ochratoxins, patulin, fumonisins,
zearalenone and trichotecenes [32,33]. In the present study, ochratoxin A (OTA) and
fumonisins B1 and B2 were quantified. These mycotoxins are commonly produced by
several Penicillium and Aspergillus spp. and Fusarium and Aspergillus spp., respectively.
Both are considered as potentially carcinogenic, immune-suppressive and nephrotoxic.
Fumonisins are further regarded as hepatotoxic [33]. The maximum permitted level of
daily OTA intake is 3 ng/g in consumed cereal products derived from unprocessed cereals
according to the EU commission regulation (EC) No 1881/2006. The mold colonies detected
in the storage trials were found to produce mycotoxins, as OTA and fumonisins B1 and
B2 were detected in isolated colonies. Nevertheless, the quantification of OTA in bread
slices was not successful as the amount of the mycotoxin was below the limit of detection.
Consequently, the level of OTA in bread slices sampled on day 10 was lower than the
limit of detection of the applied method (1.25 µg/kg). The daily intake of ochratoxin A
from cereal-based products should not exceed 3 µg/kg according to the EU commission
regulation No 1881/2006. Regarding fumonisins B1 and B2, the upper limit suggested for
the daily intake is 1000 µg/kg in maize-based foods. Using the LC-MS method described
above, no fumonisin B1 or B2 could be quantified in bread slices after 10 days of storage in
plastic bags at room temperature, even though this storage method resulted in excessive
Foods 2021, 10, 76 8 of 15

spoilage. According to this finding, the levels of fumonisins B1 and B2 were below the
suggested upper limit of daily intake, as the limits of quantification were 30.3 µg/kg and
22.7 µg/kg for fumonisins B1 and B2, respectively. It can therefore be concluded that all
breads were microbially safe and did not represent any health risk.

3.4. Bread Staling under Different Storage Conditions

Next to microbial spoilage, staling is the major reason for decreasing consumer accep-
tance with increasing storage time. Bread staling involves several different processes, such
as water redistribution and phase transitions. During baking, water is redistributed among
starch and gluten according to their gelling capacity in the crumb and is evaporated from
the crust. After the baking process, water is redistributed on a molecular and macroscopic
scale: water is exchanged between the dry crust and the moisture crumb (crust staling) and
within the crumb, where water is transferred from gluten to starch due to recrystallization
(crumb staling). In a closed system, an equilibrium would be reached after sufficient time
and a constant moisture content of crumb and crust would be set. If the crust can no longer
serve as barrier against moisture loss from the crumb, as would be the case after cutting
the bread, the bread is subjected to dehydration.
The moisture of the stored breads showed a stepwise decrease from day 0 to day 1
as the crust, serving as an intrinsic barrier, was removed and the first slice was subjected
to dehydration. The different storage methods were shown to result in different absolute
moisture losses, as presented in Table 2. Drying out was best prevented by the storage
in a plastic bag. The plastic served as an artificial barrier and hindered the air circulation
with the surrounding atmosphere. The chosen plastic wrapping had a low permeability
for water and caused the establishment of an equilibrium between crumb, crust and air
within the plastic bag. The overall decrease of moisture can be attributed to the daily
sampling procedure and the small amount of water that was absorbed from the gas within
the packaging. The storage temperature did not affect the moisture loss or the humidity in
plastic bags. The humidity inside plastic bags was 89.9% and 91.3% for room temperature
and fridge temperature, respectively. Storage inside a bread box showed the second highest
moisture loss throughout the storage time. The bread box reduced the free exchange of
air but not to the same extend as a sealed plastic bag. This was further confirmed by
the humidity inside this storage container, which was traced during the storage of the
breads and was found to be rather high in bread boxes (89.0%). The rather high humidity
can be attributed to limited air circulation and provoked the slow moisture loss of the
stored breads. The storage in a PE-layered microperforated paper bag at room temperature
resulted in the highest moisture loss. The PE-layered microperforated paper had the highest
water permeability and therefore offered the lowest barrier properties. Consequently, the
humidity inside the paper bag was the lowest found among the different storage conditions
and the closest to the humidity of the surrounding laboratory (rh = 40.5%). On the whole,
the absolute moisture loss was higher for mixed-type sourdough breads for all storing
methods. The faster drying might be attributed to the higher initial moisture content of this
bread type. As the differences in vapor pressures between the surrounding atmosphere
and the crumb were higher, a higher water transfer rate might have occurred.

Table 2. Absolute moisture loss of bread samples (crumb and crust) for different storage conditions and bread types after a
storage period of ten days (x ± STD, n = 3).

Absolute Moisture Loss of Pan Wheat Absolute Moisture Loss of Mixed-Type

Bread (%) Sourdough Bread (%)
PE-layered microperforated paper bag
10.3 ± 2.9 13.5 ± 3.3
(21.3 ± 0.4 ◦ C)
Bread box (22.0 ± 0.5 ◦ C) 7.0 ± 2.9 8.9 ± 1.3
Plastic bag (21.9 ± 0.5 ◦ C) 3.5 ± 3.1 5.7 ± 0.6
Plastic bag (8.2 ± 0.2 ◦ C) 3.1 ± 2.5 5.5 ± 1.1
 Table 2. Absolute moisture loss of bread samples (crumb and crust) for different storage conditions and bread types after
a storage period of ten days. (x ± STD, n = 3).

Absolute Moisture Loss of Pan Absolute Moisture Loss of Mixed-Type

Wheat Bread (%) Sourdough Bread (%)
Foods 2021, 10, 76
PE-layered microperforated paper bag (21.3 9 of 15
10.3 ± 2.9 13.5 ± 3.3
± 0.4 °C)
Bread box (22.0 ± 0.5 °C) 7.0 ± 2.9 8.9 ± 1.3
Plastic bag (21.9 ± 0.5 °C) 3.5 ± 3.1 5.7 ± 0.6
Plastic bag (8.2 ± 0.2 °C)
Moisture loss is known ±to2.5be
3.1 attributable to the 5.5 ±of1.1bread
firming
during the storage
period. As previously mentioned, firming is mainly caused by the retrogradation of
Moisture loss is known to be attributable to the firming of bread during the storage
amylopectin
period. Asand moisture
previously redistribution
mentioned, firmingorisloss andcaused
mainly describes theretrogradation
by the increasing hardness
of of
the crumb with increasing storage time [2]. During baking, starch
amylopectin and moisture redistribution or loss and describes the increasing hardness ofgelatinization occurs,
which therefers
crumb to withthe processstorage
increasing of thetimemelting of native
[2]. During baking,amylopectin crystals.
starch gelatinization occurs,Therefore,
which refers to the process of the melting of native amylopectin
amylopectin is in an amorphous plasticized conformation after baking. Reversing this, crystals. Therefore,
amylopectin isoccurs
recrystallization in an amorphous
during bread plasticized
storage.conformation
The side chainsafter of
baking. Reversing this,
the amylopectin molecules
recrystallization occurs during bread storage. The side chains of the amylopectin
reorganize, which results in the formation of double helices of the outer branches and
molecules reorganize, which results in the formation of double helices of the outer
increases the rigidity. Next to this, an increased formation of B-type crystalline polymorphs
branches and increases the rigidity. Next to this, an increased formation of B-type
can be observed due to the
crystalline polymorphs can retrogradation
be observed due to process. This crystal
the retrogradation structure
process. is capable of
This crystal
immobilizing more water molecules than A-type crystals [5,34] and reinforces
structure is capable of immobilizing more water molecules than A-type crystals [5,34] and the adverse
effectreinforces
of a firmer
the crumb
adversetexture.
effect of aOne measure
firmer crumb for the hardness
texture. One measure of the crumb
for the is firmness
hardness of and
the crumb is firmness and this can be accessed by texture profile analysis.
this can be accessed by texture profile analysis. As presented in Figure 1, an overall trend As presented in
can beFigure
seen1,inanwhich
overallthetrend can be
crumb seen in which
firmness the crumb
increased duringfirmness increased
the storage during
period ofthe
10 days for
storage period of 10 days for all storage conditions and bread types.
all storage conditions and bread types.

22
22
20
20
18
18
16
16
Firmness (N)

14
Firmness (N)

14
12
12
10
10
8
8
6
6
4
4
2
2
0
0
0 1 2 3 4 5 6 7 8 9 10
0 1 2 3 4 5 6 7 8 9 10
Storage time (d) Storage time (d)

(a) (b)
Figure 1. Bread firmness as a function of the storage time depending on the storage conditions applied. (a) Pan wheat
Figure 1. Bread firmness as a function of the storage time depending on the storage conditions applied. (a) Pan wheat bread.
bread. (b) Mixed-type sourdough bread. (■) Bread box at room temperature (BB), (●) PE-layered micro perforated paper
(b) Mixed-type sourdough
bag at room temperature (LP),(
bread. (▲) Bread
) plasticbox
bag at
at room temperature
room temperature (BB),
(PR) •)) PE-layered
and ((▼ micro
plastic bag fridge perforated
temperature paper
(PF). (n bag at
= 3 ± STD).(LP), (N) plastic bag at room temperature (PR) and (H) plastic bag fridge temperature (PF). (n = 3 ± STD).
room temperature

The The comparison

comparison of the
of the storage
storage methodsindicates
methods indicates an
an increased
increased firming
firming rate
ratefor
for storage
storage at fridge temperature (Figure 1). The firmness for mixed-type
at fridge temperature (Figure 1). The firmness for mixed-type sourdough bread, sourdough bread,
stored in a
stored in a plastic bag at fridge temperature, increased to 398% and for pan wheat bread
plastic bag at fridge temperature, increased to 398% and for pan wheat bread to 424%. It is
to 424%. It is commonly known that low temperatures accelerate the firming rate in starch
commonly known that low temperatures accelerate the firming rate in starch gels [35]. Gray
gels [35]. Gray and Bemiller stated that for starch gels in bread-like concentrations,
and Bemiller stated
retrogradation thatatfor
occurs starch gelsabove
temperatures in bread-like concentrations,
the glass transition temperatureretrogradation
(Tg, −5 °C) occurs
at temperatures above the glass transition temperature (Tg, − 5 ◦ C) and below the melting
and below the melting temperature (Tm, 60 °C) [20]. The recrystallization process during
temperature (Tm, 60 ◦ C)
starch retrogradation [20]. The
involves recrystallization
nucleation process
and propagation during
processes, thestarch
rates ofretrogradation
which
involves nucleation and propagation processes, the rates of which are reported to be highest
for temperatures close to Tg and Tm, respectively. Slade and Levine (1987) concluded from
their experiments that the starch retrogradation rate, as a combination of nucleation and
propagation processes, is higher for refrigerator temperatures and, therefore, the storage
of bread at refrigerator temperatures accelerates firming [34]. As expected, the storage
at room temperature resulted in considerably lower firming rates. The second highest
firming rate was found for storage in PE-layered microperforated paper bags. As this
storage method caused the highest moisture loss, as can be seen from Table 2, it is evident
that the drying out contributed to an accelerated firming of the crumb. Overall, the lowest
firming rates were found for storage in a bread box and in a sealed plastic bag. In general,
starch retrogradation bag under these two storage conditions is assumed to proceed at the
same rate as for bread stored in a layered paper bag, but drying of the crumb was limited
due to the hindered air circulation. This was also indicated by the higher humidity inside
these storage containers. In total, the lowest firming rate was observed for the plastic bag
Foods 2021, 10, 76 10 of 15

at room temperature. For this method, the firmness of the mixed-type sourdough bread
increased to 330% compared to the initial firmness and the pan wheat bread increased
to 390%. For all storage methods, the firming behavior of pan wheat bread, especially
when stored in a bread box at room temperature, was found to differ from the linear model
(storage time ≥ 5 days). This could have been caused by the occurrence of degradation
processes upon the growth of molds and yeasts.
Overall, the absolute firming rates were markedly higher for mixed-type sourdough
breads (cf. Table 3). This observation can be ascribed to the differences in crumb structure
within wheat and mixed-type breads. The denser crumb structure of the mixed -type
sourdough breads can be translated into a higher material/volume unit ratio. Jekle et al.
(2018) suggested that this causes an apparently higher firming rate due to methodical
influence [36]. As suggested by these authors, normalization can be used as tool to correct
textural measures for structural influences. The results of the normalization are presented in
Figure 2 and Table 4. After the structural correction, the normalized firming rates of mixed-
type sourdough breads were even found to be slightly lower than the normalized firming
rates of wheat bread. This might have been caused by differences in major crystallites
between rye and wheat breads. Further, the relative crystallinity of starch in rye sourdough
bread is less and was found to increase more slowly than in wheat bread [37]. Additionally,
pentosans have been reported to reduce the firmness of supplemented breads, even though
the higher moisture content of these systems was not corrected in these studies. This might
have influenced the outcomes of this study, as pentosanes bind more water and therefore
reduce the mobility of starch in the bread crumb matrix. This can probably contribute to the
decelerated recrystallization process [38]. Nevertheless, the use of mixed-type sourdough
breads might have caused only minor differences in the firming behavior.

Table 3. Absolute firming rates for different storage conditions and bread types. The results are shown in the table in terms
of the firming rate (N/d, increase in firmness per day of storage), defined as the slope of the linear model (x ± STD, n = 3).

Absolute Firming Rate of Pan Wheat Bread Absolute Firming Rate of Mixed-Type
(N/d) Sourdough Bread (N/d)
PE-layered microperforated
0.51 ± 0.07 (R2 = 0.84) 1.30 ± 0.14 (R2 = 0.90)
paper bag (21.3 ± 0.4 ◦ C)
Bread box (22.0 ± 0.5 ◦ C) 0.27 ± 0.07 (R2 = 0.61) 1.27 ± 0.12 (R2 = 0.93)
Plastic bag (21.9 ± 0.5 ◦ C) 0.29 ± 0.03 (R2 = 0.92) 0.92 ± 0.05 (R2 = 0.98)
Foods 2021, 10, x FOR PEER REVIEW 11 of 15
Plastic bag (8.2 ± 0.2 ◦ C) 0.41 ± 0.08 (R2 = 0.76) 1.38 ± 0.16 (R2 = 0.89)

6
Normalized firmness (N)

0
0 1 2 3 4 5 6 7 8 9 10
Storage time (d)

Figure
Figure2. 2.
Normalized
Normalizedbread firmness as a function
bread firmness asofathe storage time
function depending
of the storageontime
the storage
depending on the storage
conditions applied. Filled symbols and solid lines represent pan wheat bread samples, open
conditions
symbols, applied.
connected Filledlines,
by dashed symbols andmixed-type
represent solid lines represent
sourdough pansamples.
bread wheat(■bread
) Breadsamples, open symbols,
connected
box by dashed(BB),
at room temperature (●) represent
lines, mixed-type sourdough
PE-layered microperforated paper bag at bread samples. () Bread box at room
room temperature
(LP), (▲) plastic bag at room temperature (PR) and (▼) plastic bag fridge temperature (PF).
temperature (BB), (•) PE-layered microperforated paper bag at room temperature (LP), (N) plastic
Normalized firmness of the crumb over storage time was fitted using a linear model.
bag at room temperature (PR) and (H) plastic bag fridge temperature (PF). Normalized firmness of
Table 4. Firming rates for different
the crumbstorage conditions
over storage and bread
time wastypes afterusing
fitted normalization.
a linearThe results are shown in the
model.
table in terms of the firming rate, defined as the slope of the linear model (x ± STD, n = 3).

Norm. Firming Rate of Pan Wheat Norm. Firming Rate of Mixed-Type

Bread (N/d) Sourdough Bread (N/d)
PE-layered microperforated paper bag
0.46 ± 0.07 (R² = 0.84) 0.34 ± 0.04 (R² = 0.90)
(21.3 ± 0.4 °C)
Foods 2021, 10, 76 11 of 15

Table 4. Firming rates for different storage conditions and bread types after normalization. The results are shown in the table in terms
of the firming rate, defined as the slope of the linear model (x ± STD, n = 3).

Norm. Firming Rate of Pan Wheat Bread Norm. Firming Rate of Mixed-Type Sourdough
(N/d) Bread (N/d)
PE-layered microperforated
0.46 ± 0.07 (R2 = 0.84) 0.34 ± 0.04 (R2 = 0.90)
paper bag (21.3 ± 0.4 ◦ C)
Bread box (22.0 ± 0.5 ◦ C) 0.24 ± 0.06 (R2 = 0.61) 0.33 ± 0.03 (R2 = 0.93)
Plastic bag (21.9 ± 0.5 ◦ C) 0.26 ± 0.03 (R2 = 0.92) 0.24 ± 0.01 (R2 = 0.98)
Plastic bag (8.2 ± 0.2 ◦ C) 0.37 ± 0.07 (R2 = 0.76) 0.36 ± 0.04 (R2 = 0.89)

The excesses of retrogradation and drying out were both found to vary for the different
storage methods. The results indicate that the prevention of the drying of the crumb by
suitable barrier materials can retard the firming process. Further, the reduction of the
storage temperature to fridge temperature was shown to accelerate the retrogradation
process. The faster recrystallization clearly impacted the firmness of the samples and
accelerated the firming process.

4. Conclusions
Bread is a staple food consumed worldwide and has recently been reported to con-
tribute to the increasing amount of food waste. This can be attributed to staling and
spoilage processes that take place during the storage of bread. The objective of this study
was to investigate the influence of storage parameters on staling and spoilage behavior of
bread within the limits of common household methods. The design of this study aimed at
providing an objective comparison of household storage methods with opposable data to
address superficial knowledge and common prejudges. Visual inspection was shown to
represent an adequate tool for microbial safety control. Even though fungal growth was
quantifiable before it was perceived visually, no health risk was related to this as molds
do not represent a health risk on their own. Instead, mycotoxins are commonly associated
with adverse health outcomes, resulting in, e.g., carcinogenic or mutagenic effects. The pro-
duction of mycotoxins is not directly linked to the growth of fungi. Therefore, the microbial
safety of food is hard to access for consumers and misjudgment or excessive caution can
cause preventable food waste. No mycotoxins were quantified in bread slices after ten days
of storage in plastic bags at room temperature, even though this storage method resulted in
excessive spoilage. It can be concluded that disgust can prevent any foodborne health risk,
as visual appearance can prompt the avoidance of the consumption of spoiled food before
mycotoxins become detectable at quantifiable concentrations. The obtained data suggest,
therefore, that precautionary disposal of stored bread without any visually accessible mold
growth indication is not necessary. Further, we have shown that it is possible to increase
the shelf life of bread products within the limits of common household storage methods.
The fact that the proper temperatures for reducing fungal growth accelerate the firming
processes limits the possibilities for appropriate storing methods. Thus, the presented
results rule out some possibilities for prolonging shelf life. It has been demonstrated that
humid atmospheres promote spoilage. Therefore, appropriated construction of bread boxes
or packaging materials (e.g., improved water permeability or moisture scavengers) can
help to extent the shelf life of bread. In general, the correct choice of bread type for long
storage times contributes to the reduction of food waste and sourdough and rye specifically
have been shown to decelerate spoilage and staling processes. Further, sanitary conditions
are essential for the storage of bread and represent a simply accessible way to prolong the
stability of bread. The presented results may eventually lead to a more prudent use of
storage methods.
Foods 2021, 10, 76 12 of 15

Author Contributions: Conceptualization, T.A., R.K., A.B. and M.J.; methodology, T.A., R.K., M.F.,
A.N. and K.I.H.; validation, T.A., R.K., M.F., A.N. and K.I.H.; formal analysis, T.A., R.K., A.N. and
K.I.H.; investigation, T.A., R.K., M.F., A.N. and K.I.H.; writing—original draft preparation, T.A.;
writing—review and editing, R.K., A.N., K.I.H., A.B., M.J. and T.B.; visualization, T.A. and R.K.;
supervision, T.A. and R.K.; project administration, R.K., A.B.; resources, R.K., M.J. and T.B. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: We would like to thank the Westdeutscher Rundfunk, Science Department
(“Quarks”), Cologne, for taking over the costs for the bread boxes, the breads and the chemicals for
the mycotoxin analysis in order to produce a scientific film on this topic.
Foods 2021, 10, x FOR PEER REVIEW 13 of 15
Conflicts of Interest: The authors declare no conflict of interest.

Appendix A
Appendix A

(a) (b)

(c) (d)
Figure A1.
Figure A1. Exemplary
Exemplary presentation
presentation ofof the
the storage
storage setup.
setup. The
The bread loaves were
bread loaves were stored
stored in
in four
four different
different common
common storage
storage
methods. (a)
methods. (a) Bread
Bread loaf
loaf stored
stored in
in aa PE-layered
PE-layered microperforated
microperforatedpaper
paperbag
bag(21.3
(21.3±± 0.4
0.4 °C).
◦ C). (b)
(b) Mixed-type
Mixed-type sourdough
sourdough bread
bread
stored in a bread box with 13 little ventilation holes on the back side (22.0 ± 0.5 °C).
◦ (c) Pan wheat bread stored in a plastic
stored in a bread box with 13 little ventilation holes on the back side (22.0 ± 0.5 C). (c) Pan wheat bread stored in a plastic
bag at room temperature (21.9 ± 0.5 °C). (d) Pan wheat bread and mixed-type sourdough bread stored in plastic bags at
bag at room temperature (21.9 ± 0.5 ◦ C). (d) Pan wheat bread and mixed-type sourdough bread stored in plastic bags at
fridge temperature (8.2 ± 0.2 °C).  WDR.
fridge temperature (8.2 ± 0.2 ◦ C). © WDR (Westdeutscher Rundfunk).

(a) (b)
 (c) (d)
Figure A1. Exemplary presentation of the storage setup. The bread loaves were stored in four different common storage
methods. (a) Bread loaf stored in a PE-layered microperforated paper bag (21.3 ± 0.4 °C). (b) Mixed-type sourdough bread
Foods 2021, 10, 76 13 of 15
stored in a bread box with 13 little ventilation holes on the back side (22.0 ± 0.5 °C). (c) Pan wheat bread stored in a plastic
bag at room temperature (21.9 ± 0.5 °C). (d) Pan wheat bread and mixed-type sourdough bread stored in plastic bags at
fridge temperature (8.2 ± 0.2 °C).  WDR.

(a) (b)

(c)
Figure A2. Growth of molds on mixed-type sourdough bread stored in a plastic bag at room temperature. (a) Crumb after
Figure A2. Growth of molds on mixed-type sourdough bread stored in a plastic bag at room
ten days of storage without visually detectable growth of molds. (b) Top view of the bread loaf showing the first molds
temperature.
after ten days (a)Cross-contaminated
of storage. (c) Crumb after ten days of storage
mixed-type without
sourdough visually
bread slice detectable growth
after four days of molds.
of storage with (b)
a
Top view
spoiled pan wheat ofslice,
bread the bread loaf showing
highlighting the firstofmolds
the importance after
sanitary ten days
storage of storage. (c) Cross-contaminated
conditions.
mixed-type sourdough bread slice after four days of storage with a spoiled pan wheat bread slice,
Foods 2021, 10, x FOR PEER REVIEW 14 of 15
highlighting the importance of sanitary storage conditions.

(a) (b)

(c)
Figure 3. Growth
Figureof molds
A3. on pan
Growth ofwheat
moldsbread stored
on pan in a plastic
wheat bread bag at room
stored in a temperature. (a)room
plastic bag at Crumbtemperature.
after four days(a)
of
storage without visually detectable growth of molds. (b) Bottom view of the bread loaf showing the first molds after four
Crumb after four days of storage without visually detectable growth of molds. (b) Bottom view of
days of storage. (c) Crumb with excessive growth of molds at the side, top and bottom after ten days of storage.
the bread loaf showing the first molds after four days of storage. (c) Crumb with excessive growth of
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