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Food Research International
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Spoilage fungi in a bread factory in Brazil: Diversity and incidence through 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 L E I N F O
Keywords:
Moldy bread Bread-making Deterioration P. roqueforti Facilities air
A B S T R A C T
This study aimed to verify the main fungal species involved in the deterioration of different types of bread and to identify the possible sources of contamination of these products. Samples of raw materials (n= 127), en- vironmental air (n= 50) and moldy bread (n= 90) were analyzed.Aspergillus candidus,Wallemia sebi, and Penicillium roquefortiwere the predominant species in the raw materials and were isolated in samples of wheat flour, in two-thirds of the samples of rye and 62.5% of the wheatflour.Penicillium roquefortiwas isolated from all types of moldy bread analyzed andHyphopichia burtoniwas 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
Bread is a perishable food that suffers physical, chemical, sensory and microbiological changes during its storage; and fungi are the main group of microorganisms capable of spoilage this type of food (Dal Bello et al., 2007;Pitt & Hocking, 2009). Due to the high occurrence of fungi in agricultural crops used mainly asflour in the bread-making process, such as wheat and maize (Asadzadeh et al., 2014; Biro et al., 2009;
Chehri, Salleh, Yli-Mattila, Soleimani, & Yousefi, 2010;Eglezos, 2010;
Pitt & Hocking, 2009), the mycological quality of the raw material is a particular interest to the bakery industry (Pitt & Hocking, 2009) since it can limit the stability of the loaves during storage.
Fungal deterioration is strongly related to the high water activity and slightly acidic pH of this product that restricts the growth of other microorganisms. In addition, bread is an excellent source of carbohy- drates and has a porous structure that facilitates thefixation of fungal mycelia (Cauvain, 2015; Galić, Ćurić, & Gabrić, 2009;Legan, 1993;
Smith, Daifas, El-Khoury, Koukoutsis, & El-Khoury, 2004) and provides adequate support of oxygen for spores multiplication.
It is quite difficult to measure the loss of loaves attributed to fungi, which were estimated between 1 and 5% depending on the season, product formulation and processing method (Malkki & Rahua, 1978). A study conducted by Killian and Kreuger (1983) in a bakery in the
United States revealed losses of 5%, while in Brazil, and probably in other countries with a tropical climate, was estimated byFreire (2011), values on the basis of 10%.
The major genera involved in the bakery products spoilage are Penicillium (Penicillium roqueforti, Penicillium brevicompactum, and Penicillium chrysogenum),Wallemia,Aspergillus(formerlyEurotium) and other common molds, including Chrysonilia sitophila, Rhizopus and Mucor(Pitt & Hocking, 2009). Yeasts also can cause the“chalk mold”
problem, mainlySaccharomycopsis fibuligera andHyphopichia burtonii (Saranraj & Geetha, 2012).
Moreover, it is important to access and monitor the main sources of spoilage fungi through the bread processing chain. The contamination coming with raw materials, especially the diverseflours used for the production of different varieties of bread, play a relevant role in the dispersion of spores in the facilities air. Therefore, data from this study could provide subsidies for the development of methodologies to con- trol these microorganisms, reducing economic losses and promoting the increase in the shelf life of these foods.
Thus, the aim of this work was to access and identify the main fungal species involved in the deterioration of different types of bread in Brazil and try to correlate with contamination of raw materials and facilities air acting like possible sources of contamination of these products.
https://doi.org/10.1016/j.foodres.2019.108593
Received 22 March 2019; Received in revised form 23 July 2019; Accepted 26 July 2019
⁎Corresponding author.
E-mail address:[email protected](M.V. Copetti).
Available online 27 July 2019
0963-9969/ © 2019 Elsevier Ltd. All rights reserved.
T
2. Materials and methods
2.1. Sampling, isolation and identification of spoilage fungi species in raw materials and moldy bread
Raw materials (n= 127), moldy bread (n= 90) and facilities air samples (n= 50) were collected in a medium size bread industry (15 thousand bread produced per day) located in the Rio Grande do Sul State, Brazil (Latitude: −29.6914, Longitude: −53.8008 29°
41′29″South, 53 ° 48′3″West). This industry was presenting problems of early bread spoilage by fungi.
The raw materials were of different lots and corresponded to rye (n= 15), wheatflour (n= 20), wheatfiber (n= 10), linseed (n= 10), granola (n= 10), sugar (n= 10), powdered milk (n= 10), whey (n= 10), cornflour (n= 5), wholemealflour (n= 11) and oatflakes (n= 11). Moldy bread samples corresponded to: linseed bread (n= 13), wholemeal bread (n= 15), rye bread (n= 15), loaf bread (n= 24), multigrain whole bread (n= 11), and milk bread (n= 11).
These samples of packed bread get molded before the expiration date and were returned by supermarkets due visible fungal deterioration.
Collection was made in the disposal sector of the industry, before the material incineration.
Fungal isolation was performed by weighing 25 g of the sample and transferring to borosilicate reagent bottles containing 225 mL of pep- tone water (0.1%); followed by manual homogenization for approxi- mately 1 min. Subsequently, the solution wasfiltered with sterile gauze, 10−1to 10−3dilutions were prepared for raw material and up to 10−7 for analyses of moldy bread. From each dilution, 100μL were in- oculated and spread on the surface of Petri dishes containing DG18 (Dichloran 18% glycerol agar) culture medium. Plates were incubated at 25 °C for seven days and the results were expressed in colony forming units per gram of sample (CFU/g).
After the incubation period, the colonies were isolated into CYA (Czapeck yeast extract agar) for genus definition according toPitt and Hocking (2009)and, when appropriate, were subsequently directed to identification according to specific recommendations. Briefly, for gen- eralAspergillusspp. identification, isolates were three points inoculated in CYA and MEA (Malt extract agar), incubation was carried out for 7 days at 25 and 37 °C, followingKlich and Pitt (1988)recommenda- tions. Additionally, inoculation in CY20S (Czapek yeast extract agar with 20% sucrose) with incubation during 14 days at 25 °C was used for identification of xerophilic species (mainly Aspergillus section Asper- gillus, formerlyEurotium).Klich and Pitt (1988)was used for a general comparison of macroscopic and microscopic features of species, com- plemented by Visagie et al. (2014)for Aspergillus spp. from section Circumdati and Chen et al. (2017)forAspergillus section Aspergillus.
Talaromyces spp. were identified according to Yilmaz, Visagie, Houbraken, Frisvad, and Samson (2014). Finally, for identification of Penicilliumspp., basically the keys proposed byPitt (2000)andFrisvad and Samson (2004)were used for mono and biverticilliate, and ter and quaterverticilliate species, respectively. Briefly, Penicillia were three- point inoculated in different culture media [CYA, MEA (Malt Extract Agar), YES (Yeast extract sucrose agar) and CREA (Creatine sucrose agar)] and, after 7 of cultivation in different temperatures (5, 25 and 37 °C), had their diameters measured and macro and microscopic characteristics like color of verse and reverse of mycelium, exudate production, shape, size and ornamentation of conidia and conidiophore were observed. All culture media used in this study and its ingredients and proportions are listed in the Supplementary Data 1.
2.2. Determination of fungal contamination in the facilities air of the bread industry
For the analysis of the facility air, two methods were adopted: the agar printing method and the sedimentation method. The choice of collection points established in this study was because bread is a long
time exposed to the environmental air, allowing for the deposition of fungal spores on their surface. For both methods, air samples were performed forfive days in different weeks (one sampling for a week).
For the agar printing method, air samples (n= 25) were performed with a Sampl'air™(Biomérieux®), in the bread-making, oven, cooling I and II, slicing and packaging areas. During the procedure, a total of 50 L of air were sampled using DG18 culture medium. To perform the ana- lyses, 90 mm diameter Petri dishes containing about 27 mL of culture medium (Apha, 1985) were used. The plates removed from the sampler were incubated at 25 °C for seven days. The analyses were performed in duplicate in each place.
For the sedimentation method (n= 25), Petri dishes containing DG18 were arranged in duplicate at the same collection points in the industry sites for 15 min (Samson, Hoekstra, Frisvad, & Filtenborg, 2002). The plates were incubated at 25 °C for seven days and after this period, the number of colonies was counted and the predominant genera/species which grew in the plates were isolated for identification, following the steps ofSection 2.1.
2.3. Data analyses
The frequency of occurrence (FO%) of a fungal species in raw ma- terials and moldy bread was determined according to the following equation:
= ⎛
⎝
⎞
⎠× NSF
FO% TNS 100
(1) where: NFS: number of samples with the presence of species; TNS: the total number of samples.
The variation of infection (VI%) was also determined. It corresponds to the lowest and highest percentage of infection by a certain fungal species within the same sample.
Also, heatmaps were built clustering the frequency of occurrence of a fungal species in raw materials and moldy bread (Figs. 1 and 2, re- spectively) using XLSTAT-OMICS module (XLSTAT®, 2016).
Variance analysis (ANOVA) was performed. Means of frequency of occurrence of fungal contamination of raw materials, moldy bread, and environmental air were analyzed using the Scott-Knott test (P < .05).
Statistical analyses were performed using version 5.6 of SISVAR®
Software (Ferreira, 2011).
The contamination level of the environment air for the agar printed method was calculated using the following formula:
= Contamination level (CFU/m ) N
V
3
(2) where:Nis the number of microorganisms obtained by the conversion in the air sample table.Vis the volume in m3of air in the collection points.
For the sedimentation method, the fungal contamination was cal- culated according toOmeliansky (1940)using the following formula:
= −
N 5a10 (bt)4 1 (3)
where:Nis the fungal concentration (CFU/m−3),ais the number of colonies per Petri dish,bis the area of Petri dish (cm2), andt is the exposure time (min).
The isolation and identification of the fungal species as described in item 2.1.
3. Results and discussion
A total of 13 genera and 35 fungal species were isolated from raw 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 (Table 3), as well as yeasts. In raw material, the lowest mean value of contamination was found in sugar (< 10 CFU/g; P < .05) and the highest in cornflour samples (4.60 × 104CFU/g; P < .05). In moldy
bread, these values ranged from the lowest in linseed bread (2.60 × 107CFU/g; P < .05) and the highest mean value in multigrain whole bread (9 × 108CFU/g; P < .05). No statistical differences were found for facilities air from the lowest mean of contamination (1.84 × 103CFU/m3in the cooling area I) to 2.66 × 103UFC/m3in the bread-making area (by Agar printing method). Otherwise, the lowest mean of contamination was found in the oven area (3.79 × 102UFC/
m3; P < .05) and the highest in the slice and packaging area (1.25 × 104CFU/m3; P < .05) by sedimentation method.
In consonance with our data,Santos, Bernardi, Silva, Copetti, and Sant'ana (2016)determined that whole cornflour samples showed the highest mean values for fungal contamination (4.8 log CFU/g). Also, wholemeal moldy bread stored at 25 °C, showed the highest fungal count (mean of 6.2 log CFU/g). On the opposite, these authors obtained lower fungal counts in the air during pre-baking (weighing and mixer), with mean values around 2 log CFU/g. In bakeries from Brazil,Garcia, da Pia, Freire, Copetti, & Sant'Ana, (2019)showed that the higher mean fungal counts were found in the cooling and package, and exhibition areas (1.40 × 103 and 1.47 × 103CFU/g, respectively). These high counts that were found in our study in the slicing and packing area are a
cause for concern, because the fungal spores dispersed in this air can easily contaminate the freshly baked bread.
Aspergillus candidus, Aspergillus penicillioides, Wallemia sebi, and Penicillium roquefortiwere the predominant species in the raw materials and were isolated in samples of rye, wheat flour and wheat fiber (Table 1).
Aspergilli and Penicillia were predominant in both raw materials and moldy bread samples, followed byCladosporiumsp. (Tables 1 and 2). These data are in accordance with literature, once the main genera related to the deterioration of bread include Penicillium, Aspergillus sectionAspergillus (formerlyEurotium), as well as species from genus Cladosporium,MucorandRhizopus(ICMSF - International Comission on Microbiological Specifications for Foods, 2005;Pitt & Hocking, 2009).
A. candiduswas the most frequent species, isolated from > 1/3 of raw material samples (Fig. 1,Table 1), howeverP. roquefortiwas the most prevalent species related to all the types of moldy bread analyzed, followed byHyphopichia burtonii, which spoiled linseed, whole grain, rye, loaf and multigrain whole bread (Fig. 2,Table 2). It is known that the quality of ingredients and raw materials plays a fundamental role in the bread-making process, since they act as a source of contamination 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 thisfigure legend, the reader is referred to the web version of this article.)
in baking facilities. Santos et al. (2016) isolated mainly the genera Penicillium (38.2%) and Aspergillus (23.6%), followed by Aspergillus sectionAspergillus(formerlyEurotium) with 19.1% in whole-wheatflour samples. The most common species isolated from samples were Peni- cillium polonicum (16.8%), Aspergillus candidus (15.2%), Penicillium commune(8.8%) andAspergillus montevidensis (formerlyEurotium am- stelodami) (8%).
The heatmap (Fig. 1) suggests that wheatfiber and wheatflour were the main sources ofP. roquefortiamong the raw materials used in the bread-making process.H. burtoniiwas not isolated in the dilutions ac- cessed during raw materials analyses, suggesting that even this micro- organism is present in low levels; it has a competitive advantage due to the preservative resistance.
The use of preservatives has been an alternative to increase the shelf
life of bakery product, although their use does not please consumers (Membré & Kubaczka, 2000). Propionates and sorbates, and sometimes benzoates are among the most used in the baking industry (Marín et al., 2003). Despite the effectiveness of preservatives, some species of fungi show resistance, even at the maximum concentrations allowed in food.
This is the case ofP. roqueforti, which may present an extended lag phase followed by multiplication (Harris, Karahadian, & Lindsay, 1986;
Lund, Filtenborg, Westall, & Frisvad, 1996;Suhr & Nielsen, 2004), a factor that can be responsible for the predominance of this species in the deteriorated samples analyzed in this study.Moro, Lemos, Garcia, and Copetti (2019)showed the resistance ofP. roquefortiandPenicillium paneum strains isolated from moldy bread to the concentrations of propionic acid allowed for use in bread and also the resistance ofP.
roquefortito sorbic acid. According to the same study,P. roquefortiand 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.)
Table1 Fungalcontaminationofrawmaterialsusedasingredientsinbread-makingproduction. RyeWheatflourWheatfiberLinseedGranolaSugarPowderedmilkWheyCornflourWholemealflourOatfleak n=15n=20n=15n=10n=10n=10n=10n=10n=5n=11n=11 Countsaverage (CFU/g)2.7×103a2.0×103a2.6×103a1.1×103a7.7×103a1.3×101b<10c1.0×103a4.6×104d1.1×103a4.0×104d FungiFO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%)FO(%)VI(%) Alternariasp.6.66ND– 4.76–ND–ND–ND–ND–ND–ND––ND9.1ND– 1.7–ND Aspergilli100–100–100–90–20–100–0–0–100–90.1–81.8– A.candidus80ND– 62.590ND– 98.246.6ND- 10040ND– 27.2720ND−25–ND–ND–ND60ND– 24.681.1ND– 64.772.7ND- 50 A.chevalieri–ND15ND– 17.64–ND60ND– 33.3ND–ND–ND–ND60ND– 86.9536.6ND-8018.2ND– 7.14 A.flavus–ND20ND– 5.8820ND– 6.5240ND- 25ND–ND–ND–ND20ND– 9.23–ND72.7ND– 14.28 A.montevidensis–ND10ND– 7.14–ND20ND– 33.3ND–ND–ND–ND20ND- 47–ND–ND A.nigercomplex–ND–ND–ND–NDND–ND–ND–ND–ND–ND9.1ND– 16.7 A.penicillioides66.6ND– 91.725ND– 9.180ND– 30.7640ND– 12.5ND60ND-75–ND–ND60ND– 22.7–ND–ND A.pseudoglaucus20ND– 96.425ND– 4.5450ND– 26.6320ND– 4.44–ND40ND-25–ND–ND60ND– 24.6–ND36.4ND– 28.37 A.restrictus13.3ND– 14.315ND– 4.65–ND40ND– 27.27–ND–ND–ND–ND–ND40ND– 6.2536.4ND– 16.7 A.ruber–ND25ND– 35.71–ND80ND- 20–ND–ND–ND–ND40ND– 4.54–ND–ND A.sydowii–ND–ND–ND–ND–ND–ND–ND–ND–ND27.7ND– 1.7–ND A.tamarii–ND–ND–ND–NDND–ND–ND–ND–ND–ND–ND A.terreus–ND–ND6.66ND– 17.4–ND–ND–ND–ND–ND–ND–ND–ND A.versicolor–ND15ND– 27.3–ND20ND– 2.22ND10ND-50–ND–ND20ND– 15.49.1ND– 37.527.3ND– 16.7 A.wentii–ND–ND–ND–ND–ND–ND–ND–ND–ND9.1ND– 3.33–ND Cladosporiumsp.53.3ND- 43.820ND– 6.8120ND– 1.3140ND- 4520ND−5.13–ND–ND20ND- 10060ND– 9.154.5ND– 17.6454.5ND– 42.85 Eupenicillium alutaceus–ND–ND6.66ND– 2.17–ND–ND–ND–ND–ND–ND–ND–ND Fusariumsp.–ND5ND– 2.32–ND–ND–ND–ND–ND–ND40ND– 36.4–ND–ND Hyphopichiaburtonii–ND–ND–ND–ND–ND–ND–ND–ND–ND–ND–ND Nonsporulating mycelia–ND–ND–ND–ND10ND−94.8–ND–ND–ND–ND–ND–ND Mucorsp.–ND–ND–ND–NDND–ND–ND–ND–ND–ND–ND Neurosporasitophila–ND–ND–ND–ND–ND–ND–ND–ND20ND– 4.54–ND–ND Nigrosporasp.–ND–ND6.66ND– 2.17–ND–ND–ND–ND–ND–ND–ND–ND Penicillia66.6–50–46.7–40––0–0–0–20–40–36.7– P.aurantiogriseum–ND25ND– 33.36.66ND– 5.45–ND–ND–ND–ND–ND–ND–ND18.2ND– 27.3 (continuedonnextpage)
Table1(continued) RyeWheatflourWheatfiberLinseedGranolaSugarPowderedmilkWheyCornflourWholemealflourOatfleak n=15n=20n=15n=10n=10n=10n=10n=10n=5n=11n=11 Countsaverage (CFU/g)2.7×103a2.0×103a2.6×103a1.1×103a7.7×103a1.3×101b<10c1.0×103a4.6×104d1.1×103a4.0×104d FungiFO(%)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–ND6.66ND– 4.34–ND–ND–ND–ND–ND–ND–ND–ND P.citrinum–ND12.5ND– 16.712.5ND– 17.4–ND–ND–ND–ND–ND–ND–ND–ND P.commune–ND–ND–ND40ND– 6.25–ND–ND–ND–ND–ND–ND–ND P.crustosum–ND–ND–ND–ND–ND–ND–ND–ND–ND–ND–ND P.griseofulvum6.66ND– 6.25–ND–ND–ND–ND–ND–ND–ND–ND–ND–ND P.implicatum–ND–ND–ND–NDND–ND–ND–ND–ND–ND–ND P.lividum–ND–ND–ND–ND–ND–ND–ND–ND–ND9.1ND-5–ND P.paxilii–ND–ND–ND–NDND–ND–ND–ND–ND–ND–ND P.raistrickii20ND– 31.25–ND–ND–ND–ND–ND–ND–ND–ND–ND18.2ND– 28.57 P.roqueforti20ND −9.150ND– 72.146.7ND– 17.4–NDND–ND–ND–ND20ND– 12.318.8ND-20–ND Rhizopussp.–ND–ND–80ND– 56.25–ND–ND–ND–ND–ND–ND18.2ND– 27.3 Talaromyces funiculosus–ND–ND–ND–ND–ND10ND−20–ND–ND–ND–ND–ND Talaromyces islandicum–ND–ND6.66ND– 1.82–ND–ND–ND–ND–ND–ND–ND–ND Talaromyces rugulosum–ND–ND–ND–ND–ND–ND–ND–ND–ND9.1ND−5–ND Talaromyces wortmanii–ND–ND6.66ND– 2.17–ND–ND–ND–ND–ND–ND–ND–ND Wallemiasebi66.7ND- 5035ND– 6.8120ND– 2.1760ND– 36.3610ND−58.3–ND30ND−10020ND- 1220ND– 31.8545ND-5045.4ND– 9.1 Yeasts16.7ND– 33.312.5ND– 92.1453.3ND– 92.110ND- 76.930ND−10050ND-10040ND-10030ND– 98.24–ND27.3ND −6554.5ND– 28.7 FO:%ofthefrequencyofoccurrence(numberofcoloniesshowingfungalspecies/totaloffungalcontaminationinthesamples×100);ND:Notdetected(countsbelowofquantificationlimitofmethodor<10CFU/g); VI:%ofthevariationofthefungalcontaminationinthesamples;n:numberofsamples.Differentlowercaselettersinthesamelineindicatedifferencesbetweenthemeansoffrequencyofoccurrenceoffungal contaminationaccordingtoScott-Knotttest(P<.05).
H. burtoniiwere also resistant to high concentrations of acetic acid.
Regarding the airborne fungal contamination in the industry, the areas of mixture of ingredients (bread-making) and slice and packaging were the most contaminated (Table 3). Fungi from genera Clados- porium,Aspergillus(Aspergillusflavus,A. candidus) andPenicillium(Pe- nicillium citrinum, P. roqueforti) were recovered from the air of these sites. Species commonly present in the samples of raw materials (A.
candidus) and moldy bread (P. roqueforti) were also found in the pro- cessing areas.
It is well established that in bakery products processing facilities, the greatest concern is related to the airborne fungal spores since they are potential agents for the deterioration of ready-to-eat products (Legan, 1993). The available literature reports that flour contains a substantial number of fungal spores (Pitt & Hocking, 2009; Poisson, 1975;Rogers & Heseltine, 1978;Santos et al., 2016). However, these loads of spores when contaminating the bread mass are inactivated during the baking (Knight & Menlove, 1961; Garcia, da Pia, et al., 2019). Therefore, for bread to suffer fungal spoilage, it must be re- contaminated after roasting along with the cooling, slicing, or packa- ging operations (Den Aantrekker, Boom, Zwietering, & van Schothorst, 2003;Burfoot, Brown, Xu, Reavell, & Hall, 2000;Legan, 1993;Viljoen
& Von Holy, 1997). When evaluating the mycological air quality in bakeries from Brazil,Garcia, Sonnenstrahl Bregão, et al. (2019)also isolated species with deteriorative potential in the cooling and exposure areas. The deterioration occurs when fungi achieve favorable condi- tions of temperature, humidity, and aeration, which allow these mi- croorganisms to germinate and produce visible mycelium over the substrate, leading to rejection by consumers (Dagnas & Membré, 2013).
In a study carried out byLemos, Garcia, de Oliveira Mello, and Copetti (2018), moldy bakery products (bread, panettones & Easter cakes, and cakes & pies) represented 36.4% of all complaints regarding moldy foods.
The spore size also matters. Fungi with small size conidia (< 5μm) were more likely to be detected by the printing method when compared to the sedimentation method (Table 3). So, agar printing plates re- covered a wider variety of fungal species (mainlyPenicilliumsp.) when compared to sedimentation plates. Similar results were obtained by
Asefa et al. (2009), which commented that the artificial air disturbance created by the devices could affect the types of fungi recovered because particles smaller than 5μm are likely to remain suspended in the air of a facility for an extended time (Kornacki, 2014), making difficult their detection by sedimentation. On the opposite, our results (Table 3) corroborates that sedimentation is prone to selectively sample fungi of large spore size, such as Cladosporium spp. (Andon, 2006; Solomon, 1975). But, it is relative, sinceP. roqueforti, the most important spoilage agent in bread, was only recovered by the sedimentation method in this study (Table 3).
Garcia, Sonnenstrahl Bregão, et al. (2019)recommended a scale with the maximum value of fungal counts that should be present in the air to avoid bread deterioration. According to the scale proposed by the authors, in the bread industry where our study was carried out, the most places where the air was sampled, would be classified as highly contaminated, representing a high or critical concern for early spoilage occurrence.
Andrade and Salustiano (2008)observed that in the food processing areas,floor drains, ventilation systems, communication between dif- ferent rooms, spilled food, and transportation systems are important sources of aerosols. Simple cleaning processes, such as sweeping, va- cuuming and dusting, usually remove large particles, but also increase the concentration of small particles suspended in the air. Also, slicing machines, bread coolers, conveyor belts, and racks are considered sources of spoilage fungi, such asH. burtonii(Saranraj & Geetha, 2012).
So, how we can avoid the early deterioration of bread and bakery products? Once determined the source/origin of spoilage agents, sani- tization is the most practical and effective way to reduce the microbial load contaminating the processing environment, including the air, and so, controlling the losses.
The choice of sanitization agents against spoilage fungi is also very important because the efficacy of the process depends on the fungal susceptibility to the active principle and concentration of the sanitizer applied. Recently, Bernardi, Stefanello, Lemos, Garcia, and Copetti (2019)showed variability in the sensitivity to sanitizers between strains of the same species and also among different species isolated from spoiled baked goods, highlighting the importance of checking the 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 × 107a 7 × 107b 3.88 × 107a 7 × 107b 8 × 108c 9 × 108c
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
Cladosporiumsp. – 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
Sacchoromycopsisfibuligera 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).
Table3 Fungalcontaminationofenvironmentalairofthebreadindustrybytwosamplingmethods. Samplingarea BreadmakingOvenCoolingICoolingIISliceandpackaging MethodA.P.Sed.A.P.Sed.A.P.Sed.A.P.Sed.A.P.Sed. Counts(Average)(CFU/m3)Sporessize(μm)2.66×103a9.83×103b2.07×103a3.79×102b1.84×103a3.25×103a1.88×103a2.73×103a1.92×103a1.25×104b Fungi Absidiasp.3–6x Alternariasp.7–14x Aspergilluscandidus2.5–4xx Aspergilluschevalieri4–5.5x Aspergillusclavatus3–4.5xx Aspergillusflavus3–6xxxxxxxxx Aspergillusmontevidensis4–5.5xx AspergillusnigerComplex4–5xxxx Aspergillusochraceus2.5–3.5xxxxxxx Aspergillussydowi2.5–3xxx Aspergillustamarii5–8xxxxxxxxxx Aspergillusversicolor2–3.5xxx Chrysoniliasitophila6–15x Cladosporiumsp.4–9xxxxxxxxxx Fusariumsp.4–40xxxxxxx Hyphopichiaburtonii2.5–5xxxxxxxx NonsporulatingmyceliaVariousxxxxxxx Paecilomycesvariotii3–5x Penicilliumaethiopicum2.8–3.8xxx Penicilliumbilaiae2.5–3x Penicilliumcitrinum1.2–3xxxxxx Penicilliumconfertum3.2–3.7x Penicilliumglabrum3–3.5x Penicilliumjanthinellum2.3–3x Penicilliummiczynskii2.5–3x Penicilliumpalitans3.5–4.5x Penicilliumpaxilii2.2–3xxxx Penicilliumolsonii3–4x Penicilliumoxalicum3.5–7xx Penicilliumroqueforti3.5–5xxx Rhizopussp.8–20x Talaromycessp.2.5–4x Wallemiasebi1.5–4xxxxxxxx DematiaceousfungiVariousx YeastsVariousxxxxxx **A.P.:Agarprintingmethod;Sed.:Sedimentationmethod. SporesizeaccordingtoPittandHocking(2009)andSamsonetal.(2004).TheXindicatesthepresenceoffungalgeneraorspeciesbythesamplingairmethod. DifferentlowercaselettersinthesamelineindicatedifferencesbetweenthemeansoffrequencyofoccurrenceoffungalcontaminationaccordingtoScott-Knotttest(P<.05).