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Effect of packaging materials and storage conditions on the microbial quality

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DOI: 10.1007/s13197-020-04513-3

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J Food Sci Technol
https://doi.org/10.1007/s13197-020-04513-3
ORIGINAL ARTICLE
Effect of packaging materials and storage conditions
on the microbial quality of pearl millet sourdough bread
Itohan Ebunoluwa Martins1 • Toafik Akinyemi Shittu1 • Oluseye Olusegun Onabanjo2 •
Adebukola Dorcas Adesina1 • Antonio Gomes Soares4 • Pius Ifeanyi Okolie3 •
Abiodun Olajumoke Kupoluyi1 • Oluwakemi Abosede Ojo1 •
Adewale Olusegun Obadina1
Revised: 25 April 2020 / Accepted: 1 May 2020
Ó Association of Food Scientists & Technologists (India) 2020
Abstract Sourdough is one of the oldest methods of cereal
fermentation applied mainly for the sole purpose of baking
and has been proven to enhance the production of micro-
biologically safe products. This study investigated the
effect of packaging materials and storage conditions on the
microbial quality and some intrinsic parameters of the
sourdough bread. Pearl millet flour was naturally fermented
for 72 h to obtain sourdough which was used with pearl
millet flour to produce sourdough bread while the control
bread was produced with pearl millet flour and yeast. The
bread samples were packaged in low density polyethylene
and aluminium foil and stored at -5, 4, 6, 28 and 37 °C.
The total bacteria count (log cfu/g) and total fungal count
(spore/g) increased with an increase in storage temperature
and storage days. Fungi colonies isolated from stored bread
samples include species of Rhizopus, Aspergillus, Penicil-
lium and Mucor. A significant increase (p B 0.05) in the
pH and decrease in the moisture content of the bread
samples were observed as storage period and storage
temperature increased. Sourdough fermentation improved
the microbial qualities and extended the shelf life of bread.
& Itohan Ebunoluwa Martins
itohanmartins@gmail.com
1
Department of Food Science and Technology, Federal
University of Agriculture, Abeokuta, Nigeria
2
Department of Nutrition and Dietetics, Federal University of
Agriculture, Abeokuta, Nigeria
3
Biotechnology Centre, Federal University of Agriculture,
Abeokuta, Nigeria
4
Cereal Laboratory, Embrapa Food Technology, Embrapa,
Brası́lia, Brazil
Keywords Pearl millet  Sourdough bread  Fermentation 
Microbial analysis  Intrinsic parameters
Introduction
Millet is a nutritious, underutilized, and drought resistance
crop which when included in processed food can improve
its nutritional qualities (Obadina et al. 2017). Pearl millet
(Pennisetum glaucum) is the most widely cultivated millet
species and is a major source of calories, essential
micronutrients, phytochemicals, vitamins, phenolic com-
pounds, minerals and nutraceuticals, which constitutes a
major part of food security in developing countries (Ade-
biyi et al. 2016). Pearl millet grains when processed can be
eaten as ingredients in different foods and they are known
as ‘‘Nutri-cereals’’ because of their nutrients and antioxi-
dant properties (Amalraj Augustine 2015). Likewise, they
are a form of food substitute for people who are celiac and
gluten-sensitive (Annor et al. 2015). Cereal processing
contributes to an important area of the food production
chain, and its daily consumption through human diet pro-
vides essential nutrients and energy. The addition of glu-
ten-free cereals such as millet in bread increase its
nutritional benefits which includes improved cholesterol
levels, promotion of digestive health and increase in energy
levels (Adebiyi et al. 2016).
Developments in bread-making over the past years have
been both extensive and diverse, covering ingredients,
mixing processes, methods of dough development and
processing equipment, particularly in terms of automation
and in the range of bread products available to the con-
sumer (Brown 1993). Bread products play a vital role in
human nutrition as it is believed to serve as a great source
of energy and irreplaceable nutrients for the human body
123

(Dashen et al. 2016). Bread products are susceptible to
microbial spoilage, particularly mould growth which con-
fers substantial economic loss in the baking industry which
results to the short shelf life of bread (Dashen et al. 2016).
Sourdough technology varies extensively and has
become important due to its nutritional improvement,
increased shelf life, production of digestible and palat-
able food (Sakandar et al. 2018). The key features for
sourdough technology in bread making are high volume,
microbiologically safe products, good nutritional and sen-
sory properties; and prevention of bread spoilage from
bacteria and moulds (Bajerska et al. 2015). Sourdough can
be defined as an acidic sharp-tasting mixture of flour and
water for the development of bread from cereal flour,
especially wheat and rye (Sakandar et al. 2018). Sourdough
bread is a traditional product which has high nutraceutical
factors (e.g. antioxidants, vitamins, minerals) and pro-
longed shelf life due to the positive interaction of yeast and
Lactic acid bacteria (LAB) and their biochemical reactions
(Sakandar et al. 2018).
The native microorganisms present in naturally fer-
mented sourdough generally contain a complex mixture of
natural yeast and LAB which plays an important role in the
quality of sourdough bread which includes acidification,
flavour generation, mineral bioavailability and ethanol and
CO2 production, as well as bio preservation (Ogunsakin
et al. 2015). LAB has been proven to contribute positively
to different characteristics of bread such as volume, tex-
ture, and reduction in staling rate which helps to extend the
microbial shelf life of bread (Torrieri et al. 2014, Arendt
et al. 2007).
Deterioration of bread products is usually attributed to
the type of packaging materials and spoilage organisms
particularly mould (Adebowale et al. 2017). The most
popularly bread packaging materials used to be glazed
imitation parchment (GIP), impregnated on both sides with
a paraffin wax contained PE-LD and other additives, or
cellulose film (Brown 1986). Bread is usually packaged in
low density polyethylene bags with a strip of adhesive tape
used to seal the twisted end of the packaging material
(Galić et al. 2009). The aim of packaging in bread making
is to maintain the fresh condition of the bread by pre-
venting a rapid rate of drying out without providing a good
atmosphere which would promote mould growth on a
soggy crust (Balev et al. 2011). Packaging does not only
help to retain the amount and form of the required ingre-
dient and nutrients in foods but it also helps to improve the
sensory quality and colour stability by protecting the food
from degradative factors such as light, oxygen, water
vapor, moulds, yeasts, bacteria, and insects (Balev et al.
2011). There is scanty information on the packaging
materials for sourdough bread in literature, but this was
aimed at determining the effect of packaging materials and
123
J Food Sci Technol
storage conditions on the microbial quality of pearl millet
sourdough bread.
Materials and methods
Materials
Pearl millet grains used in this study was purchased from
Kuto market in Abeokuta, Ogun State, Nigeria. Packaging
materials (Low-density polyethylene of 0.920 g/cm3 den-
sity and Aluminium foil paper with a density of 2.7 g/cm3)
were also purchased from kuto market in Abeokuta, Ogun
State, Nigeria.
Methods
Preparation of pearl millet flour
The method of Deshpande and Poshadri (2011) was used in
the preparation of pearl millet flour. Pearl millets grains
were sorted to remove unwanted materials like stones,
pebbles and other foreign seeds, and washed with
portable water. The cleaned pearl millet grains were dried
in a cabinet dryer at 60 °C for 24 h to a moisture content of
about 10%. The dried grains were milled and sieved
through 500 lm aperture size. The resultant flour was
packed and sealed in polythene bags for use.
Natural fermentation of pearl millet flour
Pearl millet sourdough was prepared by mixing pearl millet
flour with water in a ratio 1:1 (weight per volume) in a
glass jar, covered lightly and allowed to ferment naturally
at room temperature for 72 h and stirred at an interval of
12 h to prevent mould growth on top of the dough
according to the modified method of (Lonner et al. 1986).
Determination of pH and TTA of pearl millet sourdough
pH and titratable acidity (TTA) of the fermenting pearl
millet sourdough were determined at 0, 12, 24, 36, 48, 60
and 72 h intervals of fermentation. 10 g of the fermenting
dough sample was homogenized in 90 ml of sterile dis-
tilled water and filtered and pH was taken using a pH meter
(Searchtech phs-3c, Zhengzhou Nanbei Instrument Equip-
ment Co., Ltd., Ningbo, Shanghai, China). 1 ml of phe-
nolphthalein indicator was introduced into 10 ml of the
homogenate from each sample and titrated against 0.1 N
Sodium Hydroxide (NaOH) for the determination of TTA
until pink colour persisted for about 10–15 s for complete
neutralization (AOAC 2005).
J Food Sci Technol
Percentage titratable acidity calculated as lactic acid:
Total tiratable acidity ðTTAÞ
Titre  Normality of base  0:009018  100
¼
Weight of sample in gram
Processing of pearl millet sourdough bread
The modified method of Edema and Sanni (2008) was
adopted for the production of the sourdough bread. The
weight of the ingredients was 15 g of sugar, 1 g of salt,
85 ml of water, 50 g of pearl millet sourdough and 100 g
of Pearl millet flour. Mixing was done using an electric
Tevion hand mixer (GT-HM-05, Globaltronics GmbH &
Co. KG, Domstrasse 19, D-20095 Hamburg, Germany) and
was mixed thoroughly for 10 min at high speed. The
mixture was scaled into a greased baking pan (7 cm
height 9 6.9 cm width 9 10 cm length), moved into the
proofing chamber (PSM-80, Guangzhou Heng Wei Kitchen
Equipment Co., Ltd., Guangzhou, Guangdong, China) for
dough maturation (2 h at 50 °C). Pearl millet bread dough
were gently transferred without agitation into an electric
baking oven (ELEM-80A, Guangzhou Heng Wei Kitchen
Equipment Co., Ltd., Guangzhou, Guangdong, China) and
baked for 1 h at 180 °C to obtain pearl millet sourdough
bread.
Control sample production of pearl millet bread
The control sample was prepared according to the modified
method of Edema and Sanni (2008) using 100% pearl
millet flour (without the inclusion of Pearl millet Sour-
dough) with the same process of pearl millet sourdough
bread stated above.
Storage Conditions and Packaging of Pearl Millet
Sourdough Bread
The pearl millet sourdough bread samples and control
samples (Pearl millet bread without sourdough) were
packaged in low-density polyethylene (LDPE) and alu-
minium foil paper. The packaged products were stored at
temperatures of -5, 4, 6, 28 and 37 °C. Analyses were
carried out daily for 4 days after production and every
other day until the evidence of visible mould growth.
Microbiological analysis of stored pearl millet sourdough
bread
Samples (10 g) of Pearl millet sourdough bread were sus-
pended in sterile 90 ml distilled water tubes and serially
diluted (ten-fold dilution). Dilutions (1 ml) of 10–3, -10–5
and 10–7 were inoculated on sterile disposable Petri dishes
using pour plate method. The plates were previously
labelled appropriately based on the different media used;
plate count agar Cm325 Oxoid (Wade Road, Basingstoke,
Hampshire, RG248PW, United Kingdom), for total bacte-
ria count and potato dextrose agar CMO 139 Oxoid (Wade
Road, Basingstoke, Hampshire, RG248PW, United King-
dom), for total mould count. The plates were then incu-
bated appropriately to allow growth of organisms; plate
count agar at 37 °C for 18–24 h and potato dextrose agar at
25 °C 72 h, total bacteria count and total fungal count were
made on the respective media (Oluwole et al. 2014).
Identification of fungal isolates from stored pearl millet
sourdough bread
Fungal isolates were identified based on the following:
Macroscopic morphology: Isolates were characterized
using the feature (colour, texture and pigment) of each
colony (Pitt and Hocking 2009).
Microscopic morphology: A drop of 95% ethanol was
placed on a sterile microscopic slide. Using a sterile
inoculating needle, a small portion of fungal growth was
gently removed from midway between the colony center
and the edge. It was ensured that both the aerial and sub-
strate (vegetative) mycelium was picked. With two dis-
secting needles, the fungus was gently teased so that it is
thinly spread out on the mounting medium. When most of
the ethanol had evaporated, a drop of lactophenol cotton
blue was added and covered with a cover slip. The fungus
was examined microscopically (Pitt and Hocking 2009).
Determination of Intrinsic parameters
Intrinsic parameters such as pH and moisture content were
carried out on pearl millet sourdough bread packaged in
different packaging materials during storage.
pH
Samples (10 g) of the bread was homogenized in 90 ml of
sterile distilled water and filtered. The pH was taken using a pH
meter (Searchtech phs-3c, Zhengzhou Nanbei Instrument
Equipment Co., Ltd., Ningbo, Shanghai, China) (AOAC 2005).
Moisture content
The moisture content of the bread samples was determined
using the method described by (AOAC 2005). Each sample
(5 g) was weighed into cleaned, dried and weighed glass
123

Petri dishes. The dishes with their content were placed
inside the hot air oven at a temperature of 105 °C for 3 h.
Thereafter the samples were cooled in the desiccators and
weighed. The dishes were taken back to the oven and
allowed to stay for 30 min. This process was continued
until a constant weight was achieved. Therefore, the per-
centage moisture content was calculated using the formula:
W1  W2 100
 increase in TTA was also reported by Amalraj Augustine
W 1
where:
W1 = Weight of sample in dish before drying.
W2 = Weight of dried sample ? dish after drying.
W = Weight of sample.
Statistical analysis
All the data obtained from the intrinsic parameters (pH and
moisture content of the pearl millet bread samples were
subjected to analysis of variance (ANOVA) to determine
the differences. The least significant test was used to detect
significant differences among the means at 5% significance
level. General linear model was used to estimate the sig-
nificant effect of packaging materials, storage temperature
and time on the microbial quality, pH and moisture content
of the bread samples.
Results and discussion
Effect of fermentation period
on the physicochemical properties of pearl millet
sourdough
Figure 1 shows the effect of fermentation time on the
physicochemical properties of pearl millet sourdough. The
pH of the sourdough ranged from 4.03–6.34 while the TTA
ranged from 0.03–0.13. A gradual and significant decline of
(p B 0.05) was observed in the pH of the sourdough while
titratable acidity increased significantly (p B 0.05) as the
Fig. 1 Effect of fermentation period on the physicochemical
properties of pearl millet sourdough. TTA Total titratable acidity
123
J Food Sci Technol
time of fermentation advanced. The steady decrease in the
pH and increase in TTA during fermentation of the pearl
millet sourdough can be attributed to the production of
organic acids such as lactic acid and acetic acids (Chavan
and Chavan 2011). Acetic acid has an anti-mould effect on
sourdough bread and antimicrobial effect on certain
microorganisms such as rope-producing Bacillus (Rosen-
quist and Hansen 1998). This trend in the decrease in pH and
(2015) which are attributed with the activities of some fer-
mentative organisms such as lactic acid bacteria and yeast in
the fermenting foods. Increase in the pH of fermenting pearl
millet sourdough over storage time was also in agreement
with the findings of Clarke et al. (2002), who reported higher
pH value for sourdough fermented with mixed strains starter
culture. Studies on sourdough have emphasized the impor-
tance of functional properties such as pH, acidity as well as
production of antimicrobial compounds in the development
of starter cultures for sourdough (Arendt et al. 2007).
Effect of packaging materials and storage conditions
on the microbial quality of pearl millet sourdough
bread
Table 1 shows the effect of packaging materials and stor-
age conditions (temperature and days) on the microbial
quality of pearl millet sourdough bread. Total bacteria
count was not detected in all the bread samples (control and
experimental) on day 0 and day 1 at all the storage tem-
perature in both packaging materials. These findings are in
line with the findings of Singh et al. (2011) who reported
similar results. Total fungal count was also not detected in
all the bread samples (control and experimental) on day 0
and day 1 at all the storage temperature in both packaging
materials. However, both total bacteria count and total
fungal count of the bread samples (control and experi-
mental) stored in both packaging materials showed
increasing trend in growth as the storage days and storage
temperature increased except in samples stored at -5 °C
with no fungal growth. This is as a result of the conversion
of water in the bread into a non-active compound during
freezing and this, along with the low temperature, retards
microorganism growth and prevent chemical and enzy-
matic deterioration of bread which encourages the growth
of mould (Majzoobi et al. 2011). The higher bacterial and
fungal population observed in the control sample stored in
both packaging materials at different temperature could be
as a result of the absence of sourdough. But fungal spoilage
is the main cause of economic loss in the baking industry
and the ability of sourdough to increase the shelf life of
bread has been widely reported (Dashen et al. 2016).
Ogunsakin et al. (2015) observed that during sourdough
process lactic acid and other metabolites which inhibit the
J Food Sci Technol

Table 1 Effect of packaging

Samples Temp (°C) TBC (Log cfu/g) TFC (spore/g)
materials and storage conditions
on the microbial quality of pearl Days of storage Days of storage
millet sourdough bread
0 1 2 3 7 14 0 1 2 3 7 14

AT 6 nil nil 3.10 3.57 5.13 nc nil nil 3.38 3.78 8.57 nc
BS nil nil 3.07 3.55 4.98 nc nil nil 3.29 3.57 8.49 nc
CR nil nil 2.41 2.97 3.20 nc nil nil 2.84 3.33 3.88 8.61
DQ nil nil 2.32 2.90 3.14 nc nil nil 2.77 3.21 3.77 8.50
EP 28 nil 3.55 3.65 nc nc nc nil nil 3.87 nc nc nc
FO nil 3.49 3.63 nc nc nc nil nil 3.63 nc nc nc
GN nil nil 3.35 nc nc nc nil nil 3.53 nc nc nc
HM nil nil 3.30 nc nc nc nil nil 3.50 nc nc nc
IL 37 nil 3.44 3.52 nc nc nc nil nil 3.86 nc nc nc
JK nil 3.42 3.50 nc nc nc nil nil 3.73 nc nc nc
KJ nil nil 3.25 5.20 nc nc nil nil 3.60 5.64 nc nc
LI nil nil 3.20 5.13 nc nc nil nil 3.45 5.56 nc nc
MH 4 nil nil 3.06 3.47 4.87 5.43 nil nil 3.21 3.57 6.47 8.85
NG nil nil 3.02 3.43 4.83 5.33 nil nil 3.18 3.43 6.39 8.83
OF nil nil 2.39 2.81 3.12 5.03 nil nil 2.51 3.22 3.68 8.54
PE nil nil 2.30 2.79 3.11 5.02 nil nil 2.44 3.18 3.55 8.53
QD -5 nil nil 2.45 3.31 3.97 4.28 nil nil nil nil nil nil
RC nil nil 2.42 3.30 3.91 4.27 nil nil nil nil nil nil
SB nil nil 2.10 2.45 3.10 3.86 nil nil nil nil nil nil
TA nil nil 2.04 2.44 3.05 3.85 nil nil nil nil nil nil
AT, control bread stored in polyethylene at 6 °C; BS, control bread stored in foil at 6 °C; CR, sourdough
bread stored in polyethylene at 6 °C; DQ, sourdough bread stored in foil at 6 °C; EP, control bread stored in
polyethylene at 28 °C; FO, control bread stored in foil at 28 °C; GN, sourdough bread stored in poly-
ethylene at 28 °C; HM, sourdough bread stored in foil at 28 °C; IL, control bread stored in polyethylene at
37 °C; JK, control bread stored in foil at 37 °C; KJ, sourdough bread stored in polyethylene at 37 °C; LI,
sourdough bread stored in foil at 37 °C; MH, control bread stored in polyethylene at 4 °C; NG, control
bread stored in foil at 4 °C; OF, sourdough bread stored in polyethylene at 4 °C; PE, sourdough bread
stored in foil at 4 °C; QD, control bread stored in polyethylene at -5 °C; RC, control bread stored in foil at
-5 °C; SB, sourdough bread stored in polyethylene at -5 °C; TA, sourdough bread stored in foil at -5 °C;
nil- no growth; nc numerous count, TBC total bacteria count; TFC total fungal count

growth of spoilage organisms and causes delay in spoilage
are produced. Control sample and pearl millet sourdough
bread stored in LDPE were shown to have a slight increase
in the microbial growth when compared to those stored in
aluminium foil. This could be attributed to the nature of the
packaging materials. Low density polyethylene is more
permeable to air, gas and water vapour and has been
reported to affect the growth and survival of microorgan-
isms in packaged foods (Ogiehor et al. 2004). Microbial
(bacterial and fungal) growth was more pronounced in the
control bread which was stored in LDPE. Despite the sig-
nificant difference in the microbial growth observed in the
bread samples (control and pearl millet sourdough bread)
stored in both packaging materials, the packaging materials
had no significant effect (p C 0.05) on the microbiological
properties of the stored bread samples. It was observed that
storage temperature and storage days showed significant
effect on the microbiological properties of the stored bread
samples (control and pearl millet sourdough bread).
Identified mould growth on pearl millet sourdough
bread stored in different packaging materials
and storage conditions
Table 2 shows the mould growth identified on pearl millet
sourdough bread and control sample during storage. No
mould colonies were identified on day 0 and day 1 for all
the bread samples (control and pearl millet sourdough
bread) stored at different temperature and packaging
materials. Mould growth were observed in all the bread
samples (control and pearl millet sourdough bread) stored
at 6, 28, 37 and 4 °C from day 2 to day 14 in both pack-
aging materials and absent in bread samples (control and

123
 J Food Sci Technol

Table 2 Identified Mould

Samples Temp (°C) Days of storage
growth on pearl millet
sourdough bread stored in 0 1 2 3 7 14
different packaging materials
and storage conditions AT 6 nil nil A, C C, B C, D, B nc
BS nil nil A, C C, B C, A, B nc
CR nil nil A, C C, D C, D A, C, D
DQ nil nil C, D C, D C, D B, C, D
EP 28 nil nil B, C, D nc nc nc
FO nil nil A, B, C nc nc nc
GN nil nil C, D nc nc nc
HM nil nil C, D nc nc nc
IL 37 nil nil A, C, D nc nc nc
JK nil nil A, B, D nc nc nc
KJ nil nil A, D B, C nc nc
LI nil nil C, D B, C nc nc
MH 4 nil nil A, D C, D C, D C, D
NG nil nil A, D C, D A, B C, D
OF nil nil A, C C, D C, D C, D
PE nil nil C, D B, C C, D C, D
QD -5 nil nil nil nil nil nil
RC nil nil nil nil nil nil
SB nil nil nil nil nil nil
TA nil nil nil nil nil nil
AT, control bread stored in polyethylene at 6 °C; BS, control bread stored in foil at 6 °C; CR, sourdough
bread stored in polyethylene at 6 °C; DQ, sourdough bread stored in foil at 6 °C; EP, control bread stored in
polyethylene at 28 °C; FO, control bread stored in foil at 28 °C; GN, sourdough bread stored in poly-
ethylene at 28 °C; HM, sourdough bread stored in foil at 28 °C; IL, control bread stored in polyethylene at
37 °C; JK, control bread stored in foil at 37 °C; KJ, sourdough bread stored in polyethylene at 37 °C; LI,
sourdough bread stored in foil at 37 °C; MH, control bread stored in polyethylene at 4 °C; NG, control
bread stored in foil at 4 °C; OF, sourdough bread stored in polyethylene at 4 °C; PE, sourdough bread
stored in foil at 4 °C; QD, control bread stored in polyethylene at -5 °C; RC, control bread stored in foil at
-5 °C; SB, sourdough bread stored in polyethylene at -5 °C; TA, sourdough bread stored in foil at -5 °C;
nc, numerous count; nil, no growth; A, Rhizopus sp; B, Aspergillus sp; C, Penicillium sp; D, Mucor sp

Table 3 Cultural and Morphological characteristics of identified moulds

Codes Cultural characteristics Morphological characteristic Fungal
isolate

A Greenish black, brownish black, purplish black,
or carbon black, spherical or columnar spore
heads
B Absence of pigmentation and smooth conidia.
White conidial heads are present
C Large fluffy white colonies almost covering the
whole surface
D Cream white/large fluffy white colonies almost
covering the whole surface
sp specie
Sporangiospores are produced inside a spherical sporangium. Rhizopus sp
Columella is present on the top of the sporangiophore. Root-like
rhizoids are found
Conidiophores arise from a foot-cell. Club shaped vesicles at top of Aspergillus
the conidiophores. Conidia are found in chains sp
Branched conidiophores with chains of conidia looks like a brush Penicillium
sp
Erect sporangiophores are formed. Sporangiophore swells ate the tip Mucor sp
to form sporangia which are globular shaped. Columella is present

123
J Food Sci Technol

Table 4 Effect of packaging materials and storage conditions on the pH of pearl millet sourdough bread
Samples Temp (°C) Days of storage
0 1 2 3 7 14

AT 6 5.75 ± 0.02b 5.78 ± 0.01d 5.83 ± 0.02f 5.85 ± 0.01 h

6.01 ± 0.00 k
6.12 ± 0.02 g

b d ef h ij g
BS 5.75 ± 0.01 5.74 ± 0.08 5.81 ± 0.01 5.84 ± 0.01 5.98 ± 0.01 6.08 ± 0.01
CR 4.67 ± 0.01a 4.73 ± 0.01bc 4.77 ± 0.02bc 4.82 ± 0.01e 4.85 ± 0.02c 4.89 ± 0.01c
a a bc b b
DQ 4.66 ± 0.02 4.68 ± 0.01 4.77 ± 0.01 4.80 ± 0.00 4.83 ± 0.01 4.86 ± 0.00bc
b e g i ij l
EP 28 5.76 ± 0.02 5.91 ± 0.01 5.94 ± 0.01 5.95 ± 0.02 5.99 ± 0.01 6.57 ± 0.08
b e g i h jk
FO 5.75 ± 0.01 5.90 ± 0.00 5.93 ± 0.01 5.94 ± 0.01 5.97 ± 0.00 6.48 ± 0.01
GN 4.67 ± 0.02a 4.74 ± 0.00c 4.81 ± 0.00d 4.84 ± 0.01e 4.90 ± 0.01e 4.94 ± 0.01d
HM 4.67 ± 0.01a 4.72 ± 0.01bc 4.79 ± 0.01c 4.83 ± 0.01e 4.88 ± 0.01d 4.92 ± 0.01d
IL 37 5.75 ± 0.01b 5.78 ± 0.02d 6.01 ± 0.00 h
6.02 ± 0.01 k
6.05 ± 0.01 l
6.28 ± 0.01i
b d g j l h
JK 5.76 ± 0.01 5.78 ± 0.0 5.95 ± 0.04 6.00 ± 0.01 6.04 ± 0.01 6.21 ± 0.01
a bc b bc e k
KJ 4.67 ± 0.01 4.73 ± 0.01 4.75 ± 0.00 4.81 ± 0.01 4.90 ± 0.01 6.49 ± 0.01
a abc a b d j
LI 4.67 ± 0.01 4.72 ± 0.00 4.71 ± 0.02 4.80 ± 0.01 4.88 ± 0.01 6.44 ± 0.01
MH 4 5.75 ± 0.01b 5.76 ± 0.01d 5.81 ± 0.01ef 5.83 ± 0.01 g
6.00 ± 0.01jk 6.02 ± 0.00f
NG 5.76 ± 0.02b 5.76 ± 0.01d 5.80 ± 0.00e 5.83 ± 0.01 g
5.99 ± 0.01ij 6.01 ± 0.01f
a ab a a a
OF 4.67 ± 0.01 4.69 ± 0.01 4.72 ± 0.00 4.76 ± 0.01 4.80 ± 0.01 4.83 ± 0.01ab
a a a a a
PE 4.66 ± 0.03 4.68 ± 0.01 4.71 ± 0.01 4.75 ± 0.00 4.80 ± 0.01 4.82 ± 0.01a
b d e f g
QD -5 5.77 ± 0.01 5.76 ± 0.01 5.79 ± 0.01 5.80 ± 0.00 5.86 ± 0.01 5.97 ± 0.03e
b d e f f
RC 5.75 ± 0.01 5.75 ± 0.02 5.79 ± 0.01 5.80 ± 0.00 5.85 ± 0.01 5.96 ± 0.02e
SB 4.66 ± 0.02a 4.69 ± 0.01a 4.72 ± 0.00a 4.75 ± 0.01a 4.79 ± 0.00a 4.82 ± 0.00a
a a a a a
TA 4.67 ± 0.01 4.68 ± 0.01 4.71 ± 0.01 4.74 ± 0.01 4.79 ± 0.00 4.82 ± 0.01a
Values are means of three replicates. Data with different superscripts within the column are significantly difference (p B 0.05)
AT, control bread stored in polyethylene at 6 °C; BS, control bread stored in foil at 6 °C; CR, sourdough bread stored in polyethylene at 6 °C;
DQ, sourdough bread stored in foil at 6 °C; EP, control bread stored in polyethylene at 28 °C; FO, control bread stored in foil at 28 °C; GN,
sourdough bread stored in polyethylene at 28 °C; HM, sourdough bread stored in foil at 28 °C; IL, control bread stored in polyethylene at 37 °C;
JK, control bread stored in foil at 37 °C; KJ, sourdough bread stored in polyethylene at 37 °C; LI, sourdough bread stored in foil at 37 °C; MH,
control bread stored in polyethylene at 4 °C; NG, control bread stored in foil at 4 °C; OF, sourdough bread stored in polyethylene at 4 °C; PE,
sourdough bread stored in foil at 4 °C; QD, control bread stored in polyethylene at -5 °C; RC, control bread stored in foil at -5 °C; SB,
sourdough bread stored in polyethylene at -5 °C; TA, sourdough bread stored in foil at -5 °C;

pearl millet sourdough bread) stored at 5 °C in both
packaging materials throughout the storage period.
Cultural and Morphological characteristics
of identified moulds
Table 3 shows the fungal isolated from control sample and
pearl millet sourdough bread stored in different packaging
materials at different storage temperature which include
species of Aspergillus, Penicillium, Rhizopus, and Mucor
as a result of the rich organic materials found in it with
sugar and carbohydrates fueling the growth of the mould
spores (Ijah et al. 2014). These organisms could have been
introduced into the production of bread at various pro-
cessing stages. This finding is in line with the study of
Daniyan and Nwokwu (2011) and Ijah et al. (2014) who
identified similar organisms in bread. These organisms
could have been responsible for the spoilage of bread.
Bread moulds like Mucor and Rhizopus are found to grow
first during bread spoilage, followed by some other fungal
like Aspergillus, Penicillium and Fusarium sp. (Raviman-
nan et al. 2016).
Effect of packaging materials and storage conditions
on the pH of pearl millet sourdough bread
This study observed significant increase in the pH of bread
samples (control and pearl millet sourdough bread) as
storage days and storage temperature increased in Table 4.
These findings agreed with the findings of Kotancilar et al.
(2009) who reported increase in the pH of vakfıkebir bread
crumb as storage days increased. Increase in the pH of
bread during storage is as a result of the moulds growth and
metabolites produced which causes staling. Katina (2005)
suggested that the acidity level of sourdough is an impor-
tant factor for crumb softness, as strong acidity (pH

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 J Food Sci Technol

Table 5 Effect of packaging materials and storage conditions on the moisture content (%) of pearl millet sourdough bread
Samples Temp (°C) Days of storage
0 1 2 3 7 14

AT 6 34.20 ± 2.90a 32.73 ± 2.87a 31.81 ± 2.12a 31.28 ± 1.68a 31.06 ± 1.69abc 30.89 ± 0.01abc
a a a a abc
BS 35.20 ± 2.90 32.84 ± 2.79 32.06 ± 1.93 31.60 ± 1.45 31.44 ± 1.53 31.09 ± 0.02abc
CR 44.61 ± 2.22b 42.58 ± 1.88bcd 42.39 ± 1.77bc 42.26 ± 1.79c 41.97 ± 1.63e 41.87 ± 1.62e
b bcd bc c e
DQ 43.57 ± 2.22 42.79 ± 1.63 42.47 ± 1.73 42.32 ± 1.80 42.18 ± 1.76 42.15 ± 1.73e
a a a a ab
EP 28 32.20 ± 2.90 31.20 ± 1.05 30.59 ± 0.37 30.52 ± 0.03 30.20 ± 0.01 29.38 ± 0.46a
a a a a abc
FO 34.20 ± 2.80 31.92 ± 0.97 31.12 ± 0.99 30.98 ± 0.01 30.74 ± 0.01 30.25 ± 0.01ab
b bcd bc bc e
GN 45.61 ± 2.32 41.74 ± 2.02 41.60 ± 1.92 41.35 ± 0.01 40.82 ± 0.01 40.54 ± 0.02e
HM 44.51 ± 2.32b 41.88 ± 2.09bcd 41.72 ± 2.01bc 41.53 ± 0.02bc 40.92 ± 0.02e 40.71 ± 0.01e
IL 37 34.20 ± 2.90a 30.54 ± 0.56a 29.86 ± 0.31a 29.77 ± 0.01a 29.43 ± 0.02a 29.12 ± 0.01a
a a a a ab
JK 34.20 ± 2.90 30.81 ± 0.66 30.23 ± 0.32 30.10 ± 0.01 29.72 ± 0.02 29.44 ± 0.01a
b b b b d
KJ 45.56 ± 2.20 38.91 ± 4.06 38.44 ± 4.46 38.30 ± 4.50 38.01 ± 0.01 37.85 ± 0.01d
b bc b b d
LI 44.61 ± 2.22 39.45 ± 3.65 38.57 ± 4.42 38.50 ± 4.44 38.12 ± 0.02 37.95 ± 0.03d
a a a a bc
MH 4 35.20 ± 2.71 33.39 ± 2.60 33.17 ± 2.72 32.54 ± 1.95 32.45 ± 2.03 32.38 ± 2.00bc
NG 34.20 ± 2.82a 33.28 ± 2.72a 33.23 ± 2.71a 32.67 ± 1.95a 32.55 ± 1.92bc 32.47 ± 1.96bc
b cd c c e
OF 46.60 ± 2.22 43.42 ± 1.93 43.12 ± 1.80 43.05 ± 1.78 42.96 ± 1.79 42.89 ± 1.76e
b cd c c e
PE 44.61 ± 2.22 43.50 ± 1.92 43.25 ± 1.78 43.14 ± 1.77 42.99 ± 1.80 42.94 ± 1.77e
a a a a c
QD -5 34.10 ± 2.90 33.63 ± 2.78 33.56 ± 2.74 33.45 ± 2.79 33.23 ± 2.83 33.04 ± 2.71c
a a a a c
RC 36.20 ± 2.80 33.71 ± 2.77 33.63 ± 2.76 33.56 ± 2.77 33.37 ± 2.74 33.26 ± 2.65c
SB 46.61 ± 2.22b 43.87 ± 1.74d 43.70 ± 1.60c 43.52 ± 1.58c 43.31 ± 1.57e 42.74 ± 1.46e
b d c c e
TA 45.61 ± 2.20 43.94 ± 1.73 43.77 ± 1.65 43.62 ± 1.59 43.41 ± 1.53 42.85 ± 1.37e
Values are means of three replicates. Data with different superscripts within the column are significantly difference (p B 0.05)
AT, control bread stored in polyethylene at 6 °C; BS, control bread stored in foil at 6 °C; CR, sourdough bread stored in polyethylene at 6 °C;
DQ, sourdough bread stored in foil at 6 °C; EP, control bread stored in polyethylene at 28 °C; FO, control bread stored in foil at 28 °C; GN,
sourdough bread stored in polyethylene at 28 °C; HM, sourdough bread stored in foil at 28 °C; IL, control bread stored in polyethylene at 37 °C;
JK, control bread stored in foil at 37 °C; KJ, sourdough bread stored in polyethylene at 37 °C; LI, sourdough bread stored in foil at 37 °C; MH,
control bread stored in polyethylene at 4 °C; NG, control bread stored in foil at 4 °C; OF, sourdough bread stored in polyethylene at 4 °C; PE,
sourdough bread stored in foil at 4 °C; QD, control bread stored in polyethylene at -5 °C; RC, control bread stored in foil at -5 °C; SB,
sourdough bread stored in polyethylene at -5 °C; TA, sourdough bread stored in foil at -5 °C;

3.6–3.8) increases softness and moderate acidity (pH
4.7–5.8) leads to a harder crumb structure. Pearl millet
sourdough bread had lower pH values when compared with
the control bread sample due to the presence of sourdough.
The increase in bread acidity in relation to increase in
sourdough concentration agrees with the findings of
(Dashen et al. 2016). Bolourian and Khodaparast (2010)
reported that there was a linear increase in the acidity (i.e.
low pH) of bread with increase in sourdough concentration.
The bread samples (control and pearl millet sourdough
bread) stored in LDPE were observed to have a slight
increase in pH when compared to bread stored in alu-
minium foil as storage days increased which may be as a
result of the permeability of the LDPE. However, pack-
aging materials, storage temperature and storage days had
no significant effect (p C 0.05) on the pH of the stored
bread samples (control and pearl millet sourdough bread).
Effect of packaging materials and storage conditions
on the moisture content of pearl millet sourdough
bread
The result of the moisture content of stored bread samples
(control and pearl millet sourdough bread) are displayed in
Table 5. A decrease in the moisture content of the pearl
millet sourdough bread and control sample were observed
as storage days and storage temperature increased. This
may be attributed to the migration of moisture from the
bread crumb to the bread crust which encourages the
growth of mould and causes subsequent increase as storage
period increases. According to Monteau et al. (2017), some
of the water lost are absorbed by the bread crust while most
of the water migrates from the crumb to atmosphere,
leading to loss of water during storage. Besbes et al. (2016)
also reported decrease in moisture content and water
activity values in breads stored at 25 °C for 23 days and at

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J Food Sci Technol
15 °C for 9 days, respectively. Pearl millet sourdough
bread and control bread samples stored in LDPE showed a
higher rate of moisture loss when compared with the bread
samples (control and pearl millet sourdough bread) stored
in aluminium foil as storage days increased. This finding is
in line with the findings of Bhise and Kaur (2014) who
observed subsequent loss of moisture content in bread
during storage. LDPE is microperforated and would allow
a faster rate of moisture loss while aluminium foil has a
property of being a better barrier to moisture. Higher
moisture retention in bread is economical and is also
required to lengthen shelf life as loss of moisture in bread
increases hardness over time. Bread moisture content-after
baking indicates the quality and can be directly correlated
to shelf life of produce (Bhise and Kaur 2014). The bread
samples (control and pearl millet sourdough bread) stored
under 37 °C and 28 °C showed a higher rate of moisture
loss than those stored at -5 °C, 4 °C and 6 °C which is as
a result of the high temperature storage. In relation to this,
packaging materials, storage temperature and storage days
had no significant effect (p C 0.05) on the moisture content
of the stored bread samples (control and pearl millet
sourdough bread).
Conclusion
In this study, acceptable bread was produced from 100%
pearl millet flour using a modified sourdough procedure
without the inclusion of chemical preservatives. The
microbiological and intrinsic properties of pearl millet
bread were evaluated. Sourdough fermentation improved
the microbial qualities and extended the shelf life of bread.
The packaging materials, storage temperature and storage
days had no significant effect (p C 0.05) on the pH and
moisture content of the stored bread samples. The pack-
aging materials had no significant effect (p C 0.05) on the
microbiological properties of the stored bread samples but
storage temperature and storage days showed significant
effect (p B 0.05) on the microbiological properties of the
stored bread samples. As a result of this, LDPE can be used
for packaging and storage of sourdough bread as it is
cheaper, flexible, strong, easy to seal, and is resistant to
moisture.
Acknowledgements This research was supported by grants from
M-BoSs (African Brazil Agricultural Innovative Market Place). The
authors also appreciate the Department of Food Science and Tech-
nology, Federal University of Agriculture, Abeokuta, for their tech-
nical support.
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