Food Research International 126 (2019) 108593

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Food Research International

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Spoilage fungi in a bread factory in Brazil: Diversity and incidence through T

the bread-making process
Marcelo Valle Garcia, Angélica Olivier Bernardi, Gilson Parussolo, Andrieli Stefanello,
⁎
Jéssica Gonçalves Lemos, Marina Venturini Copetti
Federal University of Santa Maria – UFSM, Department of Technology and Food Science, Center of Rural Sciences, 97105-900 Santa Maria, RS, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: This study aimed to verify the main fungal species involved in the deterioration of different types of bread and to
Moldy bread identify the possible sources of contamination of these products. Samples of raw materials (n = 127), en-
Bread-making vironmental air (n = 50) and moldy bread (n = 90) were analyzed. Aspergillus candidus, Wallemia sebi, and
Deterioration Penicillium roqueforti were the predominant species in the raw materials and were isolated in samples of wheat
P. roqueforti
flour, in two-thirds of the samples of rye and 62.5% of the wheat flour. Penicillium roqueforti was isolated from all
Facilities air
types of moldy bread analyzed and Hyphopichia burtoni was also present in samples of moldy wheat and rye
bread. These two species were also recovered during air sampling from baking industry facilities (cooling and
slice and package areas), which may be crucial for product contamination after baking. Hygienic measures to
reduce airborne contamination during the cooling and packaging of food should be taken to prevent the early
deterioration of bread.

1. Introduction United States revealed losses of 5%, while in Brazil, and probably in
other countries with a tropical climate, was estimated by Freire (2011),
Bread is a perishable food that suffers physical, chemical, sensory values on the basis of 10%.
and microbiological changes during its storage; and fungi are the main The major genera involved in the bakery products spoilage are
group of microorganisms capable of spoilage this type of food (Dal Bello Penicillium (Penicillium roqueforti, Penicillium brevicompactum, and
et al., 2007; Pitt & Hocking, 2009). Due to the high occurrence of fungi Penicillium chrysogenum), Wallemia, Aspergillus (formerly Eurotium) and
in agricultural crops used mainly as flour in the bread-making process, other common molds, including Chrysonilia sitophila, Rhizopus and
such as wheat and maize (Asadzadeh et al., 2014; Biro et al., 2009; Mucor (Pitt & Hocking, 2009). Yeasts also can cause the “chalk mold”
Chehri, Salleh, Yli-Mattila, Soleimani, & Yousefi, 2010; Eglezos, 2010; problem, mainly Saccharomycopsis fibuligera and Hyphopichia burtonii
Pitt & Hocking, 2009), the mycological quality of the raw material is a (Saranraj & Geetha, 2012).
particular interest to the bakery industry (Pitt & Hocking, 2009) since it Moreover, it is important to access and monitor the main sources of
can limit the stability of the loaves during storage. spoilage fungi through the bread processing chain. The contamination
Fungal deterioration is strongly related to the high water activity coming with raw materials, especially the diverse flours used for the
and slightly acidic pH of this product that restricts the growth of other production of different varieties of bread, play a relevant role in the
microorganisms. In addition, bread is an excellent source of carbohy- dispersion of spores in the facilities air. Therefore, data from this study
drates and has a porous structure that facilitates the fixation of fungal could provide subsidies for the development of methodologies to con-
mycelia (Cauvain, 2015; Galić, Ćurić, & Gabrić, 2009; Legan, 1993; trol these microorganisms, reducing economic losses and promoting the
Smith, Daifas, El-Khoury, Koukoutsis, & El-Khoury, 2004) and provides increase in the shelf life of these foods.
adequate support of oxygen for spores multiplication. Thus, the aim of this work was to access and identify the main
It is quite difficult to measure the loss of loaves attributed to fungi, fungal species involved in the deterioration of different types of bread
which were estimated between 1 and 5% depending on the season, in Brazil and try to correlate with contamination of raw materials and
product formulation and processing method (Malkki & Rahua, 1978). A facilities air acting like possible sources of contamination of these
study conducted by Killian and Kreuger (1983) in a bakery in the products.

⁎
Corresponding author.
E-mail address: mvc@smail.ufsm.br (M.V. Copetti).

https://doi.org/10.1016/j.foodres.2019.108593
Received 22 March 2019; Received in revised form 23 July 2019; Accepted 26 July 2019
Available online 27 July 2019
0963-9969/ © 2019 Elsevier Ltd. All rights reserved.
M.V. Garcia, et al. Food Research International 126 (2019) 108593

2. Materials and methods time exposed to the environmental air, allowing for the deposition of
fungal spores on their surface. For both methods, air samples were
2.1. Sampling, isolation and identification of spoilage fungi species in raw performed for five days in different weeks (one sampling for a week).
materials and moldy bread For the agar printing method, air samples (n = 25) were performed
with a Sampl'air™ (Biomérieux®), in the bread-making, oven, cooling I
Raw materials (n = 127), moldy bread (n = 90) and facilities air and II, slicing and packaging areas. During the procedure, a total of 50 L
samples (n = 50) were collected in a medium size bread industry (15 of air were sampled using DG18 culture medium. To perform the ana-
thousand bread produced per day) located in the Rio Grande do Sul lyses, 90 mm diameter Petri dishes containing about 27 mL of culture
State, Brazil (Latitude: −29.6914, Longitude: −53.8008 29° medium (Apha, 1985) were used. The plates removed from the sampler
41′29″South, 53 ° 48′3″ West). This industry was presenting problems were incubated at 25 °C for seven days. The analyses were performed in
of early bread spoilage by fungi. duplicate in each place.
The raw materials were of different lots and corresponded to rye For the sedimentation method (n = 25), Petri dishes containing
(n = 15), wheat flour (n = 20), wheat fiber (n = 10), linseed (n = 10), DG18 were arranged in duplicate at the same collection points in the
granola (n = 10), sugar (n = 10), powdered milk (n = 10), whey industry sites for 15 min (Samson, Hoekstra, Frisvad, & Filtenborg,
(n = 10), corn flour (n = 5), wholemeal flour (n = 11) and oat flakes 2002). The plates were incubated at 25 °C for seven days and after this
(n = 11). Moldy bread samples corresponded to: linseed bread period, the number of colonies was counted and the predominant
(n = 13), wholemeal bread (n = 15), rye bread (n = 15), loaf bread genera/species which grew in the plates were isolated for identification,
(n = 24), multigrain whole bread (n = 11), and milk bread (n = 11). following the steps of Section 2.1.
These samples of packed bread get molded before the expiration date
and were returned by supermarkets due visible fungal deterioration. 2.3. Data analyses
Collection was made in the disposal sector of the industry, before the
material incineration. The frequency of occurrence (FO%) of a fungal species in raw ma-
Fungal isolation was performed by weighing 25 g of the sample and terials and moldy bread was determined according to the following
transferring to borosilicate reagent bottles containing 225 mL of pep- equation:
tone water (0.1%); followed by manual homogenization for approxi-
NSF ⎞
mately 1 min. Subsequently, the solution was filtered with sterile gauze, FO% = ⎛ × 100
⎝ TNS ⎠ (1)
10−1 to 10−3 dilutions were prepared for raw material and up to 10−7
for analyses of moldy bread. From each dilution, 100 μL were in- where: NFS: number of samples with the presence of species; TNS: the
oculated and spread on the surface of Petri dishes containing DG18 total number of samples.
(Dichloran 18% glycerol agar) culture medium. Plates were incubated The variation of infection (VI%) was also determined. It corresponds
at 25 °C for seven days and the results were expressed in colony forming to the lowest and highest percentage of infection by a certain fungal
units per gram of sample (CFU/g). species within the same sample.
After the incubation period, the colonies were isolated into CYA Also, heatmaps were built clustering the frequency of occurrence of
(Czapeck yeast extract agar) for genus definition according to Pitt and a fungal species in raw materials and moldy bread (Figs. 1 and 2, re-
Hocking (2009) and, when appropriate, were subsequently directed to spectively) using XLSTAT-OMICS module (XLSTAT®, 2016).
identification according to specific recommendations. Briefly, for gen- Variance analysis (ANOVA) was performed. Means of frequency of
eral Aspergillus spp. identification, isolates were three points inoculated occurrence of fungal contamination of raw materials, moldy bread, and
in CYA and MEA (Malt extract agar), incubation was carried out for environmental air were analyzed using the Scott-Knott test (P < .05).
7 days at 25 and 37 °C, following Klich and Pitt (1988) recommenda- Statistical analyses were performed using version 5.6 of SISVAR®
tions. Additionally, inoculation in CY20S (Czapek yeast extract agar Software (Ferreira, 2011).
with 20% sucrose) with incubation during 14 days at 25 °C was used for The contamination level of the environment air for the agar printed
identification of xerophilic species (mainly Aspergillus section Asper- method was calculated using the following formula:
gillus, formerly Eurotium). Klich and Pitt (1988) was used for a general
N
comparison of macroscopic and microscopic features of species, com- Contamination level (CFU/m3) =
V (2)
plemented by Visagie et al. (2014) for Aspergillus spp. from section
Circumdati and Chen et al. (2017) for Aspergillus section Aspergillus. where: N is the number of microorganisms obtained by the conversion
Talaromyces spp. were identified according to Yilmaz, Visagie, in the air sample table. V is the volume in m3 of air in the collection
Houbraken, Frisvad, and Samson (2014). Finally, for identification of points.
Penicillium spp., basically the keys proposed by Pitt (2000) and Frisvad For the sedimentation method, the fungal contamination was cal-
and Samson (2004) were used for mono and biverticilliate, and ter and culated according to Omeliansky (1940) using the following formula:
quaterverticilliate species, respectively. Briefly, Penicillia were three-
N = 5a10 4 (bt)−1 (3)
point inoculated in different culture media [CYA, MEA (Malt Extract
−3
Agar), YES (Yeast extract sucrose agar) and CREA (Creatine sucrose where: N is the fungal concentration (CFU/m ), a is the number of
agar)] and, after 7 of cultivation in different temperatures (5, 25 and colonies per Petri dish, b is the area of Petri dish (cm2), and t is the
37 °C), had their diameters measured and macro and microscopic exposure time (min).
characteristics like color of verse and reverse of mycelium, exudate The isolation and identification of the fungal species as described in
production, shape, size and ornamentation of conidia and conidiophore item 2.1.
were observed. All culture media used in this study and its ingredients
and proportions are listed in the Supplementary Data 1. 3. Results and discussion

2.2. Determination of fungal contamination in the facilities air of the bread A total of 13 genera and 35 fungal species were isolated from raw
industry materials (Table 1) and six genera and 14 species from moldy bread
(Table 2). In the facilities air, 12 genera and 26 species were recovered
For the analysis of the facility air, two methods were adopted: the (Table 3), as well as yeasts. In raw material, the lowest mean value of
agar printing method and the sedimentation method. The choice of contamination was found in sugar (< 10 CFU/g; P < .05) and the
collection points established in this study was because bread is a long highest in corn flour samples (4.60 × 104 CFU/g; P < .05). In moldy

2
M.V. Garcia, et al. Food Research International 126 (2019) 108593

Fig. 1. Cluster analysis of the frequency of occurrence of fungal species in raw materials used in bread-making. The relative frequency of occurrence for each species/
genus is colored in shades of red (high relative frequency) to yellow (low relative frequency), as shown in the color key. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)

bread, these values ranged from the lowest in linseed bread cause for concern, because the fungal spores dispersed in this air can
(2.60 × 107 CFU/g; P < .05) and the highest mean value in multigrain easily contaminate the freshly baked bread.
whole bread (9 × 108 CFU/g; P < .05). No statistical differences were Aspergillus candidus, Aspergillus penicillioides, Wallemia sebi, and
found for facilities air from the lowest mean of contamination Penicillium roqueforti were the predominant species in the raw materials
(1.84 × 103 CFU/m3 in the cooling area I) to 2.66 × 103 UFC/m3 in the and were isolated in samples of rye, wheat flour and wheat fiber
bread-making area (by Agar printing method). Otherwise, the lowest (Table 1).
mean of contamination was found in the oven area (3.79 × 102 UFC/ Aspergilli and Penicillia were predominant in both raw materials
m3; P < .05) and the highest in the slice and packaging area and moldy bread samples, followed by Cladosporium sp. (Tables 1 and
(1.25 × 104 CFU/m3; P < .05) by sedimentation method. 2). These data are in accordance with literature, once the main genera
In consonance with our data, Santos, Bernardi, Silva, Copetti, and related to the deterioration of bread include Penicillium, Aspergillus
Sant'ana (2016) determined that whole corn flour samples showed the section Aspergillus (formerly Eurotium), as well as species from genus
highest mean values for fungal contamination (4.8 log CFU/g). Also, Cladosporium, Mucor and Rhizopus (ICMSF - International Comission on
wholemeal moldy bread stored at 25 °C, showed the highest fungal Microbiological Specifications for Foods, 2005; Pitt & Hocking, 2009).
count (mean of 6.2 log CFU/g). On the opposite, these authors obtained A. candidus was the most frequent species, isolated from > 1/3 of
lower fungal counts in the air during pre-baking (weighing and mixer), raw material samples (Fig. 1, Table 1), however P. roqueforti was the
with mean values around 2 log CFU/g. In bakeries from Brazil, Garcia, most prevalent species related to all the types of moldy bread analyzed,
da Pia, Freire, Copetti, & Sant'Ana, (2019) showed that the higher mean followed by Hyphopichia burtonii, which spoiled linseed, whole grain,
fungal counts were found in the cooling and package, and exhibition rye, loaf and multigrain whole bread (Fig. 2, Table 2). It is known that
areas (1.40 × 103 and 1.47 × 103 CFU/g, respectively). These high the quality of ingredients and raw materials plays a fundamental role in
counts that were found in our study in the slicing and packing area are a the bread-making process, since they act as a source of contamination

3
M.V. Garcia, et al. Food Research International 126 (2019) 108593

Fig. 2. Cluster analysis of the frequency of occurrence of fungal species in analyzed moldy bread. The relative frequency of occurrence for each species/genus is
colored in shades of red (high relative frequency) to yellow (low relative frequency), as shown in the color key. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)

in baking facilities. Santos et al. (2016) isolated mainly the genera life of bakery product, although their use does not please consumers
Penicillium (38.2%) and Aspergillus (23.6%), followed by Aspergillus (Membré & Kubaczka, 2000). Propionates and sorbates, and sometimes
section Aspergillus (formerly Eurotium) with 19.1% in whole-wheat flour benzoates are among the most used in the baking industry (Marín et al.,
samples. The most common species isolated from samples were Peni- 2003). Despite the effectiveness of preservatives, some species of fungi
cillium polonicum (16.8%), Aspergillus candidus (15.2%), Penicillium show resistance, even at the maximum concentrations allowed in food.
commune (8.8%) and Aspergillus montevidensis (formerly Eurotium am- This is the case of P. roqueforti, which may present an extended lag
stelodami) (8%). phase followed by multiplication (Harris, Karahadian, & Lindsay, 1986;
The heatmap (Fig. 1) suggests that wheat fiber and wheat flour were Lund, Filtenborg, Westall, & Frisvad, 1996; Suhr & Nielsen, 2004), a
the main sources of P. roqueforti among the raw materials used in the factor that can be responsible for the predominance of this species in
bread-making process. H. burtonii was not isolated in the dilutions ac- the deteriorated samples analyzed in this study. Moro, Lemos, Garcia,
cessed during raw materials analyses, suggesting that even this micro- and Copetti (2019) showed the resistance of P. roqueforti and Penicillium
organism is present in low levels; it has a competitive advantage due to paneum strains isolated from moldy bread to the concentrations of
the preservative resistance. propionic acid allowed for use in bread and also the resistance of P.
The use of preservatives has been an alternative to increase the shelf roqueforti to sorbic acid. According to the same study, P. roqueforti and

4
 Table 1
Fungal contamination of raw materials used as ingredients in bread-making production.
Rye Wheat flour Wheat fiber Linseed Granola Sugar Powdered milk Whey Corn flour Wholemeal flour Oat fleak
M.V. Garcia, et al.

n = 15 n = 20 n = 15 n = 10 n = 10 n = 10 n = 10 n = 10 n=5 n = 11 n = 11

3 3 3 3 3 1 3 4 3
Counts average 2.7 × 10 a 2.0 × 10 a 2.6 × 10 a 1.1 × 10 a 7.7 × 10 a 1.3 × 10 b < 10 c 1.0 × 10 a 4.6 × 10 d 1.1 × 10 a 4.0 × 104 d
(CFU/g)

Fungi FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%)

Alternaria sp. 6.66 ND – – ND – ND – ND – ND – ND – ND – – ND 9.1 ND – – ND

4.76 1.7
Aspergilli 100 – 100 – 100 – 90 – 20 – 100 – 0 – 0 – 100 – 90.1 – 81.8 –
A. candidus 80 ND – 90 ND – 46.6 ND - 40 ND – 20 ND −25 – ND – ND – ND 60 ND – 81.1 ND – 72.7 ND -
62.5 98.2 100 27.27 24.6 64.7 50
A. chevalieri – ND 15 ND – – ND 60 ND – ND – ND – ND – ND 60 ND – 36.6 ND - 80 18.2 ND –
17.64 33.3 86.95 7.14
A. flavus – ND 20 ND – 20 ND – 40 ND - ND – ND – ND – ND 20 ND – – ND 72.7 ND –
5.88 6.52 25 9.23 14.28
A. montevidensis – ND 10 ND – – ND 20 ND – ND – ND – ND – ND 20 ND - – ND – ND
7.14 33.3 47
A. niger complex – ND – ND – ND – ND ND – ND – ND – ND – ND – ND 9.1 ND –
16.7
A. penicillioides 66.6 ND – 25 ND – 80 ND – 40 ND – ND 60 ND - 75 – ND – ND 60 ND – – ND – ND
91.7 9.1 30.76 12.5 22.7
A. pseudoglaucus 20 ND – 25 ND – 50 ND – 20 ND – – ND 40 ND - 25 – ND – ND 60 ND – – ND 36.4 ND –
96.4 4.54 26.63 4.44 24.6 28.37
A. restrictus 13.3 ND – 15 ND – – ND 40 ND – – ND – ND – ND – ND – ND 40 ND – 36.4 ND –

5
14.3 4.65 27.27 6.25 16.7
A. ruber – ND 25 ND – – ND 80 ND - – ND – ND – ND – ND 40 ND – – ND – ND
35.71 20 4.54
A. sydowii – ND – ND – ND – ND – ND – ND – ND – ND – ND 27.7 ND – – ND
1.7
A. tamarii – ND – ND – ND – ND ND – ND – ND – ND – ND – ND – ND
A. terreus – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
17.4
A. versicolor – ND 15 ND – – ND 20 ND – ND 10 ND - 50 – ND – ND 20 ND – 9.1 ND – 27.3 ND –
27.3 2.22 15.4 37.5 16.7
A. wentii – ND – ND – ND – ND – ND – ND – ND – ND – ND 9.1 ND – – ND
3.33
Cladosporium sp. 53.3 ND - 20 ND – 20 ND – 40 ND - 20 ND −5.13 – ND – ND 20 ND - 60 ND – 54.5 ND – 54.5 ND –
43.8 6.81 1.31 45 100 9.1 17.64 42.85
Eupenicillium – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
alutaceus 2.17
Fusarium sp. – ND 5 ND – – ND – ND – ND – ND – ND – ND 40 ND – – ND – ND
2.32 36.4
Hyphopichia burtonii – ND – ND – ND – ND – ND – ND – ND – ND – ND – ND – ND
Non sporulating – ND – ND – ND – ND 10 ND −94.8 – ND – ND – ND – ND – ND – ND
mycelia
Mucor sp. – ND – ND – ND – ND ND – ND – ND – ND – ND – ND – ND
Neurospora sitophila – ND – ND – ND – ND – ND – ND – ND – ND 20 ND – – ND – ND
4.54
Nigrospora sp. – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
2.17
Penicillia 66.6 – 50 – 46.7 – 40 – – 0 – 0 – 0 – 20 – 40 – 36.7 –
P. aurantiogriseum – ND 25 ND – 6.66 ND – – ND – ND – ND – ND – ND – ND – ND 18.2 ND –
33.3 5.45 27.3
(continued on next page)
Food Research International 126 (2019) 108593
 Table 1 (continued)

Rye Wheat flour Wheat fiber Linseed Granola Sugar Powdered milk Whey Corn flour Wholemeal flour Oat fleak

n = 15 n = 20 n = 15 n = 10 n = 10 n = 10 n = 10 n = 10 n=5 n = 11 n = 11
M.V. Garcia, et al.

Counts average 2.7 × 103 a 2.0 × 103 a 2.6 × 103 a 1.1 × 103 a 7.7 × 103 a 1.3 × 101 b < 10 c 1.0 × 103 a 4.6 × 104 d 1.1 × 103 a 4.0 × 104 d
(CFU/g)

Fungi FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%)

P. lividum – ND – ND – – ND – ND – ND – ND – ND – ND – ND – ND
P. brevicompactum – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
4.34
P. citrinum – ND 12.5 ND – 12.5 ND – – ND – ND – ND – ND – ND – ND – ND – ND
16.7 17.4
P. commune – ND – ND – ND 40 ND – – ND – ND – ND – ND – ND – ND – ND
6.25
P. crustosum – ND – ND – ND – ND – ND – ND – ND – ND – ND – ND – ND
P. griseofulvum 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND – ND – ND
6.25
P. implicatum – ND – ND – ND – ND ND – ND – ND – ND – ND – ND – ND
P. lividum – ND – ND – ND – ND – ND – ND – ND – ND – ND 9.1 ND - 5 – ND
P. paxilii – ND – ND – ND – ND ND – ND – ND – ND – ND – ND – ND
P. raistrickii 20 ND – – ND – ND – ND – ND – ND – ND – ND – ND – ND 18.2 ND –
31.25 28.57
P. roqueforti 20 ND 50 ND – 46.7 ND – – ND ND – ND – ND – ND 20 ND – 18.8 ND - 20 – ND
−9.1 72.1 17.4 12.3
Rhizopus sp. – ND – ND – 80 ND – – ND – ND – ND – ND – ND – ND 18.2 ND –
56.25 27.3

6
Talaromyces – ND – ND – ND – ND – ND 10 ND −20 – ND – ND – ND – ND – ND
funiculosus
Talaromyces – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
islandicum 1.82
Talaromyces – ND – ND – ND – ND – ND – ND – ND – ND – ND 9.1 ND −5 – ND
rugulosum
Talaromyces – ND – ND 6.66 ND – – ND – ND – ND – ND – ND – ND – ND – ND
wortmanii 2.17
Wallemia sebi 66.7 ND - 35 ND – 20 ND – 60 ND – 10 ND −58.3 – ND 30 ND −100 20 ND - 20 ND – 545 ND - 50 45.4 ND –
50 6.81 2.17 36.36 12 31.8 9.1
Yeasts 16.7 ND – 12.5 ND – 53.3 ND – 10 ND- 30 ND −100 50 ND - 100 40 ND-100 30 ND – – ND 27.3 ND 54.5 ND –
33.3 92.14 92.1 76.9 98.24 −65 28.7

FO: % of the frequency of occurrence (number of colonies showing fungal species/total of fungal contamination in the samples × 100); ND: Not detected (counts below of quantification limit of method or < 10 CFU/g);
VI: % of the variation of the fungal contamination in the samples; n: number of samples. Different lowercase letters in the same line indicate differences between the means of frequency of occurrence of fungal
contamination according to Scott-Knott test (P < .05).
Food Research International 126 (2019) 108593
M.V. Garcia, et al. Food Research International 126 (2019) 108593

Table 2
Fungal contamination on moldy bread.
Linseed bread Wholemeal bread Rye bread Loaf bread Milk bread Multigrain whole bread

n = 13 n = 15 n = 15 n = 24 n = 12 n = 11

Counts average (CFU/g) 2.60 × 107 a 7 × 107 b 3.88 × 107 a 7 × 107 b 8 × 108 c 9 × 108 c

Fungi FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%) FO (%) VI (%)

Aspergilli 27.3 – 8.3 – 25 – 10 – 22.2 – 50 –

Aspergillus chevalieri – ND 12.5 ND – 68.7 12.5 ND – 12.5 – ND 11.1 ND - 2,5 – ND
Aspergillus restrictus – ND – ND 12.5 ND – 38.1 – ND – ND – ND
Aspergillus pseudoglaucus 33.3 ND- 46.7 – ND – ND 10 ND – 30.15 22.2 ND - 68,6 50 ND - 90
Aspergillus tamarii – ND – ND – ND – ND 11.1 ND - 17,1 – ND
Aspergillus versicolor 16.7 ND – 13.3 – ND – ND – ND – ND 33.3 ND - 10
Cladosporium sp. – ND 33.3 ND - 50 25 ND - 50 10 ND - 10 – ND – ND
Hyphopichia burtonii 50 ND - 95 37.5 ND - 100 50 ND - 100 40 ND - 100 – ND 25 ND – 70.6
Penicillia 100 – 66.7 – 62.5 – 50 – 77.8 – 87.5 –
Penicillium brevicompactum – ND – ND – ND 10 ND – 38.1 – ND – ND
Penicillium citrinum 9.1 10.31 – ND – ND – ND – ND – ND
Penicillium corylophilum – ND – ND – ND – ND 11.1 ND - 2,5 – ND
Penicillium crustosum 33.3 ND – 5.4 – ND – ND – ND – ND – ND
Penicilliium lividum 16.7 ND – 2.4 – ND – ND – ND – ND – ND
Penicillium roqueforti 81.8 ND - 100 66.6 ND - 100 37.5 ND - 100 70 ND - 100 100 ND - 100 87.5 ND- 100
Sacchoromycopsis fibuligera 16.6 ND - 20 – ND – ND – ND 11.1 ND - 32,3 – ND
Wallemia sebi 16.7 ND – 15.8 8.3 ND – 7.1 – ND – ND 11.1 ND - 8,3 16.7 ND – 0.9
Yeasts – ND – ND – ND – ND – ND 16.7 ND- 91.1

FO: % of the frequency of occurrence (number of colonies showing fungal species/total of fungal contamination in the samples × 100); ND: Not detected (counts
below of quantification limit of method or < 10 CFU/g); VI: % of the variation of the fungal contamination in the samples; n: number of samples. Different lowercase
letters in the same line indicate differences between the means of frequency of occurrence of fungal contamination according to Scott-Knott test (P < .05).

H. burtonii were also resistant to high concentrations of acetic acid. Asefa et al. (2009), which commented that the artificial air disturbance
Regarding the airborne fungal contamination in the industry, the created by the devices could affect the types of fungi recovered because
areas of mixture of ingredients (bread-making) and slice and packaging particles smaller than 5 μm are likely to remain suspended in the air of a
were the most contaminated (Table 3). Fungi from genera Clados- facility for an extended time (Kornacki, 2014), making difficult their
porium, Aspergillus (Aspergillus flavus, A. candidus) and Penicillium (Pe- detection by sedimentation. On the opposite, our results (Table 3)
nicillium citrinum, P. roqueforti) were recovered from the air of these corroborates that sedimentation is prone to selectively sample fungi of
sites. Species commonly present in the samples of raw materials (A. large spore size, such as Cladosporium spp. (Andon, 2006; Solomon,
candidus) and moldy bread (P. roqueforti) were also found in the pro- 1975). But, it is relative, since P. roqueforti, the most important spoilage
cessing areas. agent in bread, was only recovered by the sedimentation method in this
It is well established that in bakery products processing facilities, study (Table 3).
the greatest concern is related to the airborne fungal spores since they Garcia, Sonnenstrahl Bregão, et al. (2019) recommended a scale
are potential agents for the deterioration of ready-to-eat products with the maximum value of fungal counts that should be present in the
(Legan, 1993). The available literature reports that flour contains a air to avoid bread deterioration. According to the scale proposed by the
substantial number of fungal spores (Pitt & Hocking, 2009; Poisson, authors, in the bread industry where our study was carried out, the
1975; Rogers & Heseltine, 1978; Santos et al., 2016). However, these most places where the air was sampled, would be classified as highly
loads of spores when contaminating the bread mass are inactivated contaminated, representing a high or critical concern for early spoilage
during the baking (Knight & Menlove, 1961; Garcia, da Pia, et al., occurrence.
2019). Therefore, for bread to suffer fungal spoilage, it must be re- Andrade and Salustiano (2008) observed that in the food processing
contaminated after roasting along with the cooling, slicing, or packa- areas, floor drains, ventilation systems, communication between dif-
ging operations (Den Aantrekker, Boom, Zwietering, & van Schothorst, ferent rooms, spilled food, and transportation systems are important
2003; Burfoot, Brown, Xu, Reavell, & Hall, 2000; Legan, 1993; Viljoen sources of aerosols. Simple cleaning processes, such as sweeping, va-
& Von Holy, 1997). When evaluating the mycological air quality in cuuming and dusting, usually remove large particles, but also increase
bakeries from Brazil, Garcia, Sonnenstrahl Bregão, et al. (2019) also the concentration of small particles suspended in the air. Also, slicing
isolated species with deteriorative potential in the cooling and exposure machines, bread coolers, conveyor belts, and racks are considered
areas. The deterioration occurs when fungi achieve favorable condi- sources of spoilage fungi, such as H. burtonii (Saranraj & Geetha, 2012).
tions of temperature, humidity, and aeration, which allow these mi- So, how we can avoid the early deterioration of bread and bakery
croorganisms to germinate and produce visible mycelium over the products? Once determined the source/origin of spoilage agents, sani-
substrate, leading to rejection by consumers (Dagnas & Membré, 2013). tization is the most practical and effective way to reduce the microbial
In a study carried out by Lemos, Garcia, de Oliveira Mello, and Copetti load contaminating the processing environment, including the air, and
(2018), moldy bakery products (bread, panettones & Easter cakes, and so, controlling the losses.
cakes & pies) represented 36.4% of all complaints regarding moldy The choice of sanitization agents against spoilage fungi is also very
foods. important because the efficacy of the process depends on the fungal
The spore size also matters. Fungi with small size conidia (< 5 μm) susceptibility to the active principle and concentration of the sanitizer
were more likely to be detected by the printing method when compared applied. Recently, Bernardi, Stefanello, Lemos, Garcia, and Copetti
to the sedimentation method (Table 3). So, agar printing plates re- (2019) showed variability in the sensitivity to sanitizers between strains
covered a wider variety of fungal species (mainly Penicillium sp.) when of the same species and also among different species isolated from
compared to sedimentation plates. Similar results were obtained by spoiled baked goods, highlighting the importance of checking the

7
 Table 3
M.V. Garcia, et al.

Fungal contamination of environmental air of the bread industry by two sampling methods.
Sampling area

Bread making Oven Cooling I Cooling II Slice and packaging

Method A.P. Sed. A.P. Sed. A.P. Sed. A.P. Sed. A.P. Sed.

Counts (Average) (CFU/m3) Spores size (μm) 2.66 × 103 a 9.83 × 103 b 2.07 × 103 a 3.79 × 102 b 1.84 × 103 a 3.25 × 103 a 1.88 × 103 a 2.73 × 103 a 1.92 × 103 a 1.25 × 104 b

Fungi
Absidia sp. 3–6 x
Alternaria sp. 7–14 x
Aspergillus candidus 2.5–4 x x
Aspergillus chevalieri 4–5.5 x
Aspergillus clavatus 3–4.5 x x
Aspergillus flavus 3–6 x x x x x x x x x
Aspergillus montevidensis 4–5.5 x x
Aspergillus niger Complex 4–5 x x x x
Aspergillus ochraceus 2.5–3.5 x x x x x x x
Aspergillus sydowi 2.5–3 x x x
Aspergillus tamarii 5–8 x x x x x x x x x x
Aspergillus versicolor 2–3.5 x x x
Chrysonilia sitophila 6–15 x
Cladosporium sp. 4–9 x x x x x x x x x x

8
Fusarium sp. 4–40 x x x x x x x
Hyphopichia burtonii 2.5–5 x x x x x x x x
Non sporulating mycelia Various x x x x x x x
Paecilomyces variotii 3–5 x
Penicillium aethiopicum 2.8–3.8 x x x
Penicillium bilaiae 2.5–3 x
Penicillium citrinum 1.2–3 x x x x x x
Penicillium confertum 3.2–3.7 x
Penicillium glabrum 3–3.5 x
Penicillium janthinellum 2.3–3 x
Penicillium miczynskii 2.5–3 x
Penicillium palitans 3.5–4.5 x
Penicillium paxilii 2.2–3 x x x x
Penicillium olsonii 3–4 x
Penicillium oxalicum 3.5–7 x x
Penicillium roqueforti 3.5–5 x x x
Rhizopus sp. 8–20 x
Talaromyces sp. 2.5–4 x
Wallemia sebi 1.5–4 x x x x x x x x
Dematiaceous fungi Various x
Yeasts Various x x x x x x

* * A.P.: Agar printing method; Sed.: Sedimentation method.

Spore size according to Pitt and Hocking (2009) and Samson et al. (2004). The X indicates the presence of fungal genera or species by the sampling air method.
Different lowercase letters in the same line indicate differences between the means of frequency of occurrence of fungal contamination according to Scott-Knott test (P < .05).
Food Research International 126 (2019) 108593
M.V. Garcia, et al. Food Research International 126 (2019) 108593

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& Copetti, M. V. (2019). Incidence of spoilage fungi in the air of bakeries with dif-
ferent hygienic status. International Journal of Food Microbiology, 290, 254–261.
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