Growth Characterization of Mycotoxigenic Molds Isolated from Bread Flour Consumed in Algeria

Cereals are more widely consumed worldwide, have significant nutritional power and constitute the basis of the human diet. In Algeria, among the most consumed cereals in several forms, soft wheat is the most consumed. Flour is the main product made from soft wheat and is mainly used for the preparation of bread. In Algeria, 49 million baguettes are consumed every day, making them the leading bread-consuming country in the world (Belhocine, 2010).

In most cases, cereal production is ensured by a single harvest during the year, which requires storage, which can sometimes occur under poorly controlled conditions and can affect the presence and growth of several mycotoxigenic fungi during the storage of soft wheat until bread is produced (Chukwudi et al., 2021; Dutta et al., 2918; Okioma et al., 2021; Lavkor et al., 2019).

Wheat flour is characterized by lower (0.8 – 0.87) water activity (Feillet, 2000) and is produced without any processing for removing microorganisms. The fungi could survive at low aw, as reported by Dooletkedieva (2010); Graves et Hasseltine (1966); Deligeorgakis et al., (2023) and Kichu et al., (2019). With the climate change observed in recent years, fungal contamination is certainly increasing, as reported by Rezazadeh et al., (2013) for Aspergillus flavus, Aspergillus ochraceus, Aspergillus versicolor, Penicillium citrinum, Penicilliusm citreoviiride, Penicillium cyclopium, Penicillium martensii, Penicillium patulum and Penicillium pubertum. Some of these species are toxigenic, such as Aspergillus flavus, Aspergillus parasiticus, Penicillium expansum and Penicillium crustosum (Afssa, 2006).

These mycotoxigenic fungi can produce toxic secondary metabolites called mycotoxins (Awuchi et al., 2021). Mycotoxins are hazardous for consumer health and can cause great economic losses (Bhat and Vasanthi, 2003). Due to the heat stability of mycotoxins, their residue can persist in bread after heating and can be eaten by consumers.

Even if the dose is nontoxic, repeated exposure to a low dose of mycotoxins in bread is likely to cause chronic poisoning. In the long-term, mycotoxins cause harmful effects on the cardiovascular, respiratory and digestive systems, as well as on the nervous system and the urinary system (Omotayo et al., 2019). It also induces immuno-suppressive, genotoxic, carcinogenic and teratogenic mutagenic effects (Bennet and Klich, 2003; Afssa, 2006). The objective of our work was to study the contamination of soft wheat flour used for bread making in western Algeria by mycotoxigenic fungi and to model its growth ability.

The current research was carried out at University of Tlemcen from November 2020 to Jully 2023.
 
Sampling
 
A total of 241 samples of flour used for bread making in bakeries in different regions of western Algeria (Tlemcen, Ain Temouchent and Sidi Belabbes) were taken for research on mycotoxinogenic fungi. The sampling zones were selected using the areal method. It consists of randomly surrounding areas on the map. Then, each area was visited to spot bakeries and removed from the dough mixer, which was filled only with flour. A mass of 50 g of flour was taken and then placed in a sterile bag. All samples were coded as shown in Table 1.


 
Mycological analyses
 
The flour analysis was carried out following the procedure described by the Algerian standard of the Official Journal No. 52/2015, which describes the horizontal method for the enumeration of yeasts and molds by counting the colonies in the products, including those for which the water activity is less than or equal to 0.95 (aw).
 
Sample preparation
 
The procedure consisted of suspending a mass of 1 g of each sample in 9 mL of TSE. This mixture constitutes the first dilution, which will be used to prepare the range of dilutions. The mixture was rigorously shaken between each sample.
 
Counting of mold colonies
 
A volume of 100 µL of each dilution was spread on the surface of a Petri dish containing a Dichloran agar medium supplemented with 18% glycerol (enzymatic digest of casein: 5 g, bacterial agar: 15 g, chloramphenicol: 0.1 g, D-glucose: 10 g, magnesium sulfate: 0.5, potassium dihydrogenphosphate: 1 g, dichloromethane (2,6 dichlora-4-nitro-aniline): 0.002 g, glycerol: 220 g), a solution of trace elements (ZnSO₄, CuSO₄) and an oxytetracycline antibiotic at a rate of 50 mg/L to prevent the development of bacteria (Hocking and Pitt, 1980). The inoculated Petri dishes were incubated at 25^oC ± 1^oC for five to seven days.
 
Purification of mold colonies
 
The single-spore method described by Noman et al., (2018) was used to purify the isolates. Each colony mold developed on the previous medium (dichloran agar) was removed and then transplanted individually using a sterile platinum loop in the center of a Petri dish containing PDA medium, after which the cultures were incubated for five days at 25^oC.
 
Identification of mold species 
 
The identification of the mold genera of the obtained isolates was based only on microscopic observation of the colonies from culture on PDA media. Macroscopic observation proved difficult due to variability in the macroscopic aspects of colonies. In fact, for some fungi strains, the colony changes the color aspect according to the medium composition, others one affected by the light and time incubation. Thus, the identification was based on microscopic observation of isolates is based essentially on the morphological appearance and characteristics of the thallus and spores. Pitt and Hocking (1997) guide were used to identified the fungi.

The smear preparation consisted of adhering a fraction of the colony using a piece of tape and then sticking it on a slide containing a drop of mounting liquid (cotton blue). Microscopic examination was carried out using an optical microscope at x 10, x 40 and x 100 magnification.

The use of microscopic identification does not prevent the macroscopic aspect from being ignored. isolates (the color, size and texture of the colonies, the color of the reverse, the appearance of the thallus and the presence of a pigment).
 
Study of mycotoxin production
 
Detection of Mycotoxins by Thin-Layer Chromatography was carried out according to the procedure described by Scott et al., (1970). Thirty isolates of mold were inoculated with a colony fragment in vials containing 50 mL of yeast extract sucrose (YES) broth enriched with Tri B vitamins (B1, B6, B12). The cultures were incubated at 25^oC for 14 days.

After incubation, the cultures were filtered using Wattman No. 01 type filter paper. Then, the obtained filtrate was mixed with 100 mL of chloroform, stirred for 10 min and then decanted using a separatory funnel. The operation was repeated by successively adding 50 mL and 30 mL of chloroform to the recovered aqueous phase, after which the chloroform phase was concentrated by evaporation under vacuum using a rotary evaporator. After drying, the extract was returned to a glass vial enclosed by parafilm for analysis by thin layer chromatography.

Estimation of the growth parameters of mold isolates
 
For each tested mold isolate, a 2 mm diameter fragment was inoculated in the center of Petri dishes containing PDA media. During incubation, the colony diameter was measured with a ruler (Baranyi et al., 1993). From the start of growth, the colony diameter was 2 mm. The growth was monitored twice daily for one week.

The primary growth model of Baranyi et al., (1993) was used to fit the growth of the tested isolate. The growth parameters (growth rate µT°C: µmax and lag phase : ›) of each mold isolate were estimated by equation 1.
 

                         ...1

              ....2

                                                                                                                  

Where,
y₀ = Initial colony radius at time 0.
y = Colony radius at time (t : days) 
𝜆 (days) = Latency time at t (days).
A = A variable, depending the t, µmax and 𝜆, used only to simplified the equation.
 
Statistical analysis
 
The ANOVA analysis was carried out using (SPSS) computer program, ver. 22.0 and the significance among the samples was calculated at P≤0.05.

Prevalence of contamination
 
Mycological analyses of all flour samples taken from different bakeries in the regions of Ain Témouchent, Tle-mcen and Sidi Belabbes revealed the presence of molds. These results show a dominance of molds, which reached an overall contamination rate of 97.93%. These results are similar to the results obtained by Graves and Hesseltine (1966), Moreau (1970), Halt et al., (2004) and Al-Defiery and Merjan (2015).

The region of Ain Témouchent was the most contaminated region, followed by the regions of Tlemcen and Sidi Belabbes, which were less contaminated, with contamination rates of 100%, 98.25% and 93.33%, respec-tively. The ANOVA analysis showed a significant difference between two regions of Ain Temouchent and Tlemcen against Sidi Belabbes. This difference may be related to high moisture of Ain Temouchent and Tlemcen compared with Sidi Belabbes (Saïfouni and Bellatreche, 2020). Mohammed et al., (2015) reported a correlation accounting of molds and moisture content in wheat flour samples. According also to Saric et al., (2008), cereals and cereal products can be contaminated with molds in any phase of a processing cycle: in fields, during harvest, storage, processing, transport and over a period between production and consummation.
 
Mold count
 
The enumeration results obtained by this study are shown in Table 1. This indicates a total average mold load of 5.71 log (cfu/g). This concentration exceeds the threshold “M = 4 log” required by the Algerian regulations of the decree of July 2, 2017.

The results obtained show a variation in concentration between the studied regions of 3.35 log (cfu/g) for samples collected from Ain Témouchent, 9.32 log (cfu/g) for Tlemcen and 7.16 log (cfu/g) for Sidi belabbes. ANOVA revealed a significant difference between the different analyzed brands. Thiss variability is probably due in particular to the brand processing and quality of the raw material and flour used as well as the processing, preparation and storage conditions, such as the environment, humidity, time and temperature.

The contamination results obtained in this study are greater than the contamination (1.5 to 1.7 log cfu/g), (<2.3 log cfu/g) and (<2 log cfu/g) reported by Al-Defiery and Merjan (2015), Tahani et al., (2008) and Zebiri (2020), respectively. Only the results for samples from the Ain Témouchent region are in accordance with the results (0 - 4.2 log cfu/g) reported by Halt et al., (2004). The fungal concentrations (cfu) and contamination frequencies are summarized in Table 1.

The high concentrations of mould contamination could be result of inadequate sanitary measures performed during especially transport and storage of raw material, processing.
 
Mold identification
 
The results of macroscopic examinations of the colonies are characterized by variability in appearance depending on the type of mold. The main results are illustrated in Table 2. Indeed, as an example of an isolate identified as Penicillium sp. (Fig 1), on PDA media, colonies of a bluish green color with a white outline, downy to powdery, cottony, velvety, with brown granules, were observed.





Microscopic examination of the pure isolate was carried out by observation at x 40 magnification. The results reported in Table 2 reveal 11 different genera of mold: Penicillium sp., Cladosporium sp., Microsporum sp., Aspergillus sp., Auerobasidium sp., Exopiala sp., Mucor sp., Trichothecium sp., Trichoderma sp., Streptomyces sp. and Trichosporon sp.

Among the genera identified in this study, Penicillium sp., Aspergillus sp., Cladosporium sp. and Mucor sp. have also been identified by several researchers (Halt et al., 2004; Rezazadeh et al., 2013; Al-Defiery and Merjan, 2015). Other researchers have isolated only Aspergillus sp. and Penicillium sp. (Tahani et al., 2008). According to Pelhate (1982) and Berthier and Valla (1998), the genera Penicillium sp. and Aspergillus sp. are considered storage contaminants.

In this study, the list of genes identified was not exhaustive. That is, the absence of unidentified genera does not negate their presence. Indeed, the presence and prevalence are significantly linked to the number of samples tested, the quantity and number of samples, the sampling method and the analysis method and procedure.

The different identified genera had markedly variable contamination frequencies from all tested samples; the most frequent were Penicillium spp. (69.29%), followed by Cladosporium spp. (56.85%), Microsporum spp. (51.04%) and Aspergillus spp. (34.85%). In addition, other less impor-tant genera were also present, namely, Auerobasidium sp. (24.07%), Exopiala sp. (21.58%), Mucor sp. (17.43%), Trichothecium sp. (9.13%), Trichoderma sp. (8.71%), Streptomyces sp. (2.07%) and Trichosporon (0.41%).
 
Revelation of mycotoxin-producing strains by TLC
 
Thin-layer chromatographic separation makes it possible to confirm the results previously obtained on CEA media. Chromatographic separation via thin-layer TLC makes it possible to separate the extracts from the secondary metabolites found at the wheat flour substrate level by organic solvents and the detection of the extracts under UV radiation at a wavelength equal to 365 nm allows the detection of blue and green stains, which confirms the presence of mycotoxins. The toxin that appears with blue fluorescence is likely aflatoxin B1, which is produced mainly by Aspergillus. The Penicillium genus likely produces ochratoxin A, which emits green fluorescence under UV light. Betina (1985), Atanda et al., (2013) were used this technique to detect mycotoxins exhibiting blue fluorescence and blue-green fluorescence under UV light (365 nm) during the identification of mycotoxin production by the tested isolates.

Thin layer chromatography reveals the production of mycotoxins in the YES medium. The same previously tested strains were tested on YES media. Additionally, only the genera Aspergillus spp. and Penicillium sp. showed mycotoxin production.

Indeed, Aspergillus and Penicillium are storage fungi and are known to be producers of mycotoxins (Lozada, 1995). Several studies, such as Rahman et al., (2015) and Shareef et al., (2010), have also reported the production of mycotoxins by these two genera. This technique is quick, easy to perform and allows for the simultaneous processing of multiple samples (Marin et al., 2013).

Otherwise, several works reported the presence of mycotoxins in wheat flours that ranged from 0.7~74.9% and their average contamination levels in wheat flours (0.2~57.6 µg kg^-1) (Zhou et al., 2022). These mycotoxins are produced by mould.
 
Modeling of mold growth
 
Applying predictive microbiology to quantify the cfu concentration is difficult (Gibson and Hocking, 1997) because filamentous fungi are not unicellular. They form a mycelium whose weight, except at the early stage of growth, does not increase exponentially. Additionally, it is not possible to divide the mycelium into individual cells. Therefore, the colony forming unit (CFU) quantification method can only be used to enumerate spores (Vindeløv and Arneborg, 2002).

In general, the kinetic models used are those based on measuring colony diameter, a simple technique for obtaining data. The growth of molds on solid substrates under optimal conditions and in the absence of limiting factors generally follows a model consisting of a latency time (λ_ToC) and a linear growth phase (to estimate µ_ToC). Under unfavorable conditions, a stationary phase can appear when fungi stop growing (Gibson et al., 1994 and Vo et al., 2024).

The growth kinetics were adjusted using the Baranyi et al., (1993) model as reported by Dantigny et al., (2005). It shows a good fit, with R² values between 0.90 and 1. The isolates tested showed different growth capacities, as indicated by the latency time (λ_ToC: day) and growth rate (µ_ToC: day^-1). Indeed, the latency times and µ_ToC range between [0.01 to 2 days] and [0.17 to 8 days^-1], respectively. The results show that the growth parameters depend on the strain (Table 3). Indeed, variability has been observed within the same genera. Furthermore, the mycotoxigenic molds (Aspergillus spp. and Penicillium spp.) presented intermediate latency times (λ_ToC) and growth rates (µ_ToC) compared to those of the other isolates tested (Table 3).



The growth kkinetics obeys first-order kinetics characteristic of microbial growth, similar to that of bacteria, having different growth phases: latency time (λ_ToC), exponential phase (µ_ToC) and stationary phase.

Throughout the food chain, from the field to the consumer’s plate, molds are likely to develop and produce toxins, especially if ecological conditions (humidity and temperature) are favorable. Contamination of food or seeds can occur before or during the storage of baking flour. Most toxic molds grow in foods with low water activity by producing mycotoxins.

The results showed an ability to grow in wheat flour. Heenan et al., (1998) reported a growth of some Aspergillus sp., Penicillium sp. and Fusarium in wheat flour samples after three months of storage period. Among them there are A. ochreaceus, P. virdicatum, P. cyclopium, P. verricossium, A. niger, A. mellus and A. carbonarius (Heenan et al., 1998).

Others authors reported a growth of this fungi in bread with mycotoxins contamination (Ollinger et al., 2024). the consumption of small amount of mycotoxins daily and frequently could accumulates in liver the uptake of these causing serious damage and liver cancer (Halt et al., 2004).

Fungal contamination of baking flour is inevitable and the flour can grow during product storage. Among these fungi, some genera can produce mycotoxins that are involved in consumer health damage. The main results revealed the presence of a large broad range of genera, including mycobacterium fungi (Penicillium and Aspergillus), that report a production of mycotoxins laboratory medium. Repeated consumption of these mycotoxins can cause damage to public health. To this end, this work has outlined a risk assessment as a perspective.

The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.