1Graduate Program in Industrial Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand;
2Department of Industrial Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand;
3Center of Excellence in Materials Science and Technology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand;
4Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
This research investigated the optimal conditions of heat and chemical treatment for elimination of aflatoxin. NaOH showed the highest inhibition percentage (75.44%) of Aspergillus flavus growth that was isolated from low-grade maize. Low-grade maize contaminated with A. flavus was treated with NaOH by varying three factors in three levels: NaOH concentration (0, 2.5 and 5% w/v), temperature (25, 50 and 75°C) and time (24, 48 and 72 h). Aflatoxin was removed from low-grade maize after sprinkling with 5% w/v NaOH at 50°C for 24 h that reduced aflatoxin B1 content to 4.25 µg/kg with 60.35% reduction from initial value. The use of NaOH solution with simple heat influenced the reduction in fungal contamination and elimination of aflatoxin B1.
Key words: aflatoxin, aflatoxigenic fungi, chemical concentration, fungal inhibition, maize, temperature
*Corresponding Author: Suparin Chaiklangmuang, Department of Industrial Chemistry; Center of Excellence in Materials Science and Technology; Materials Science Research Center, Faculty of Science, Chiang Mai University, -Chiang Mai, 50200 Thailand. Email: [email protected]
Received: 11 November 2022; Accepted: 8 May 2023; Published: 1 July 2023
© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)
Aflatoxins are the main group of mycotoxins commonly produced by Aspergillus species such as Aspergillus flavus, A. nomius and A. parasiticus. Agricultural commodities, food and feed that enrich in carbohydrates such as maize, rice, wheat and barley are susceptible to aflatoxin contamination due to carbohydrates acting as carbon source for fungal growth. The entry of fungus in crops causes contamination and leads to the production of aflatoxin. These toxic compounds are produced under suitable humidity and temperature conditions. The main types of aflatoxins are AFB1, AFB2, AFG1 and AFG2, which are found in common contaminations. AFB1 is the most toxic and is classified under primary group of carcinogen compounds as the first group, with liver being the primary target organ, and it affects humans and animals and also lead to economic losses. AFB1 is a stable compound that cannot be destroyed during most of the food processes (Eslami et al., 2015; Heshmati et al., 2021; Kumar et al., 2021; Nejad et al., 2014a).
Aflatoxins are rarely eliminated in food-chain process due to their heat-stable property; they are highly resistant to heat treatments. Therefore, simple drying process cannot significantly reduce their contamination rates in stored grains. However, heat treatment for a prolonged time seems to have a beneficial effect on the decontamination (Pankaj et al., 2018; Peng et al., 2018; Sipos et al., 2021). Several studies have widely used chemical and physical treatments to minimize the risk of aflatoxin contamination in feeds and foods, which usually focussed on the inhibition of mycelia spore development and the consequent elimination of aflatoxins (Tian and Chun, 2017). The effects of heating methods on aflatoxin detoxification in many crops depend on temperature and time such as in steam under pressure and dry roasting (Jalili, 2016; Peng et al., 2018). Kumar et al. (2021) reported that AFB1 contents in maize were reduced by 80% when the seeds were exposed to sunlight for 10–12 h. The usage of chemical additives on contaminated foods has also become a popular choice (Rushing and Selim, 2019). Several chemicals, acids, alkalis and oxidizing agents have proved to mitigate the aflatoxigenic fungal growth and aflatoxin production when used in appropriate quantity (Kumar et al., 2021). Some food preservatives such as propionic, benzoic and boric acids, and sodium acetate also inhibit the growth of A. flavus thereby eliminating aflatoxin. The chemical treatment converts AFB1 into degradation products and thereby reduces the toxicity of AFB1 molecules (Aiko and Mehta, 2015). Recent researches have found that physical and chemical methods can be applied safely with high efficiency for the elimination of aflatoxin, which makes feeds or foods much more acceptable to consumers (Sipos et al., 2021).
This study aimed at the elimination of aflatoxin B1 that contaminated low-grade maize using both physical and chemical methods. We investigated the actions of seven kinds of chemicals, namely sodium hydroxide (NaOH), ammonium carbonate [(NH4)2CO3], calcium hydroxide [Ca(OH)2], sodium sulfite (Na2SO3), potassium hydroxide (KOH), sodium chloride (NaCl) and hydrogen peroxide (H2O2) on aflatoxigenic fungal growth. The elimination of aflatoxin B1 in low-grade maize was carried by optimising chemical concentration (0, 2.5, and 5% w/v), temperature (25, 50, 75°C) and time (24, 48, 72 h) through sprinkling method.
Low-grade maize (Zea mays L.) grains with high moisture content, discolouration and kernel damage (kernels were broken and incomplete) were obtained from Phatananikom Kaset Ltd. Company, Phrae, Thailand. Maize grains were ground and sieved to around to 2 mm particle size. The ground maize was kept in double polypropylene plastic bags and stored at room temperature (25–30°C) until use (Boonma et al., 2018).
The aflatoxigenic fungi were isolated from low-grade maize sample by using pour plating method. One gram low-grade maize was added to 9 mL sterile peptone (1% w/v) solution and performed serial dilution up to 10−6. One mL of each serial dilution solutions was placed on to three sterile petri dishes (replicas), and molten potato dextrose agar (PDA) was poured over the inoculum. All plates were rotated manually and incubated at 30°C for 7 days. The differential mycelial fungi that could be observed by naked eyes were isolated by transferring on to fresh PDA plates under sterile conditions and incubated at 30°C for 7 days (Sanders, 2012). The morphological features of each isolated fungus were observed under the microscope 40X (Olympus, Japan). The identification of different forms of fungi were confirmed by comparing with the published data or descriptive key (Barnett and Hunter, 1987). Moreover, one of the fungal strains that was expected to produce aflatoxin was selected to confirm the strains using nucleotide sequence analysis. Molecular identification was carried out by DNA barcoding using the ITS region sequencing and compared to those in the database using NCBI-BLAST.
The growth of aflatoxigenic fungi isolated from low-grade maize through the above procedure was inhibited by seven kinds of chemical reagents, namely sodium hydroxide (NaOH), ammonium carbonate [(NH4)2CO3], calcium hydroxide [Ca(OH)2], sodium sulfite (Na2SO3), potassium hydroxide (KOH), sodium chloride (NaCl) and hydrogen peroxide (H2O2). The fungal growth inhibitions were determined using three agar plating methods.
The antifungal activity of chemical solutions was determined by examining the fungal hyphal extension using the method by Ali et al. (2020). The 7-days old, 6 mm plug of old aflatoxigenic fungi was placed on the center of the PDA medium supplemented with 10% w/v of the seven chemical solutions. The PDA plate without chemical solution was used as control. The experiments replicate in the three individual experiments and all test plates were incubated at 30°C. Fungal growth or hyphal extension was performed by measuring the diameter of fungal mycelial every day for 7 days. The inhibition percentages of each chemical were calculated from the fungal growth diameter on the chemical-supplemented plates and that of the control plate (PDA) using following equation:
% Inhibition of fungal mycelial growth = (C−T)/C × 100
Where, C is the average of the three replicates of hyphal diameter (mm) of control, and T is the average of three replicates of hyphal diameter (mm) of chemical-treated plate.
The hole-plate diffusion on PDA growth medium followed the method by Nejad et al. (2014) with some modification. 100 µL of fungal suspension (106 spore/mL in 0.85% sterile saline solution) was spread on the PDA plates. After resting for 10 min, 6 mm diameter holes of uniform distance were punched on to the plates, and 20 µL each of seven sterile chemical solutions (10% w/v) and water (as control sample) were filled in each hole. The antifungal capacity was conducted after 7 days at 30°C by observing the clear zone around the holes.
The disc diffusion method with some modifications was followed by Gaziano et al. (2018). 100 µL of fungal suspension (106 spores/mL in 0.85% sterile saline solution) was spread on the PDA plates. After resting them for 10 min, sterile Whatman No.1 filter papers of 6 mm diameter, soaked in 10% w/v of each of the seven chemical solutions, were placed on the fungal-spread PDA at uniform distance. The antifungal capacity was determined after 7 days at 30°C by observing the clear zone around the filter paper discs.
The chemical reagent that mostly inhibited the growth of aflatoxigenic fungi from the chemical selection methods discussed above was used for the elimination of aflatoxin B1 in low-grade maize, along with heat and chemical. Low-grade maize (15 g) and fungal spores (106 spores/mL) were added to a polypropylene plastic bag (15 × 22 cm) and pluged with cotton wool. The procedure followed the method described by Thanaboripat et al. (1997) with some modifications. Each experiment sample was treated with selected chemical through sprinkling method based on a three-level, three-variable of 10 mL chemical concentration (0, 2.5 and 5% w/v), temperature (25, 50 and 75°C) and time (24, 48 and 72 h). The experimental design was generated with 33 full-factorial design with the experimental data, which were analysed from the means of triplicate determinations.
The optimum operational conditions were determined based on the moisture content and number of contaminated fungi. The optimal data was then determined using statistical analysis. Selected samples from the experiments were analysed for determining the optimal conditions that are able to reduce the aflatoxin B1 content.
Treated low-grade maize samples (3–5 g) were weighed to four decimal places with a moisture can of known weight. The sample in the moisture can was heated in a hot air oven at 105°C for at least 3–6 h and left at room temperature in a desiccator until a constant weight was attained (AOAC, 2000).
The number of contaminated fungi in the low-grade maize sample was determined using pour plating technique, same as that of the method used in the isolation of fungi. The plates were incubated at 30°C for 24–48 h in duplication in an inverted position. The number of fungi was counted and was recorded in the range of 30–300 cells in the spores-forming plate.
Aflatoxin B1 was analysed using a high-performance liquid chromatography-fluorescence detector (HPLC-FD), a modification of the method reported by AOAC 991.31 (2002). 25 g of ground maize samples, 5 g NaCl and 125 mL methanol and water (7:3, v/v) were added to blender and homogenised at high speed for 2 min. The mixture was filtered through pre-folded paper. 15 mL of the filtrate was then added into 125 mL glass-stopper Erlenmeyer flask and mixed with 30 mL bromine stopper in water. The solution was filtered through glass microfibre paper ≤ 30 min before affinity column chromatography; 10 mL of second filtrate (equivalent to 1 g test portion) was passed through IAC AflatestTM at a flow rate of 1.0 mL/min. The AFs were subsequently eluted from the column with 2.0 mL of methanol and collected in a glass vial, and 1.5 mL of pure water was then added to the vial. 50 µL of this test eluted solution was injected to the HPLC-FLD (Alilent Technologies, USA, series 1100). The mobile phase used during HPLC analysis was methanol, acetonitrile and water, and the flow rate was 1 mL/min. The quantity of aflatoxin in each eluate injected was determined from standard curves.
The statistical analysis of data and mathematical calculations of models generated were performed by employing the Design Expert software version 7.0.0 (Stat-Ease). The optimal data was analysed by means of Response Surface Methodology (RSM), using analysis of variance and significant F-values indicated a confidence level of 95%. A probability test of P < 0.05 was used to estimate the statistical significance of variation in the observed responses using analysis of variance (ANOVA).
Low-grade maize isolated fungi appeared in four different morphologies and colours of fungal mycelium determined by visual observation (agar plate and microscope, 40X) on 7 days of incubation as shown in Figure 1. Different morphologies dispersed from filamentous cluster (Figures 1a and 1c) and spherical pellets (Figures 1b and 1d), which consisted of aggregated hyphal structures. Different colours were observed as follows: black–green, black, green and white mycelium (Figures 1a, 1b, 1c and 1d, respectively), and fungal hyphae were presented in a light-colour that turned dark with age. In Figure 1a, the morphology of black–green mycelium revealed dark green and black powdery mycelium on agar plate, and the microscopic photograph showed the arrangement of various round conidia. In Figure 1b, black fungus dispersed and grew as fluffy mycelium and showed single round conidia. In Figure 1c, green-coloured mycelium colony developed, which formed a prominent growth ring with powdery appearance, and the colour changed from yellow to olive green with white border encircling covered the sporulating mycelia as presented on the agar plate. The colonies showed round conidia from brush arrangement of phialospores-like globose vesicles under the microscope. Morphology of the green fungus on agar plate was similar to the reproductive spore of A. flavus as described by Afzal et al. (2013), who reported that the morphological identification of A. flavus from the soil of Larkana district (Sidh, Pakistan) showed yellow to green or dark green colony. The microscopic features of green fungus were same as the morphology of A. flavus from groundnut kernels according to the study by Okayo et al. (2020). The round conidia from brush arrangement of phialospores were biseriate with radial phialides. In Figure 1d, white mycelial fungus showed deep cottony texture and became more full cotton with age. The colony of white mycelia showed sickle-shaped macroconidia under the microscope.
Figure 1. Morphological and characteristic features of mycelia of fungi isolated from low-grade maize. (A) Black-green; (B) Black; (C) Green; (D) White.
The morphology of green fungus was most likely colony and microscopic feature of A. flavus was aflatoxin--forming fungi according to the morphological identification, which was similar to that of previous study findings. The typical morphology of A. flavus are dark yellow conidia which then transforms to a dominant olive-green and surrounds with white circle. Their borders of colonies are generally plain and flat, which are raised from the centre. Under the microscope, the conidiophores of A. flavus are rough and bearing vesicles, the vesicle appears as globose to sub-globose with numerous uniseriate and biseriate columnar Aspergillus heads (Khan et al., 2020; Makhlouf et al., 2017; Thathana et al., 2017). Therefore, the isolated green fungus was selected to confirm the classification into species level by ITS region of nucleotide sequence analysis. The nuclear--encoded rDNA of isolated green fungi investigated significant alignments of 97–99% with Aspergillus sp. as in the species of A. flavus. According to the morphology and nucleotide sequence analyses, green fungus was identified as A. flavus, which normally contaminate agricultural products during storage and produce aflatoxin B1 as reported by Jaibangyang et al. (2021). Thus, the isolated green fungus from low-grade maize was used in the further experiments of chemicals selection for aflatoxin B1 elimination by heat and chemical treatments.
Seven chemicals, NaOH, (NH4)2CO3, Ca(OH)2, Na2SO3, KOH, NaCl and H2O2, were investigated and one among them was selected as the chemical for heat and chemical treatments. The selection of chemical was performed through three agar plate methods.
The growths of green fungi on seven chemical--supplemented agar plates through fungal hyphal extension method were different, which resulted in different fungal growth inhibition percentages during the incubation as shown in Figure 2. The percentage of inhibitions were less than 50% on the supplemented plates of Na2SO3, NaCl, KOH, Ca(OH)2 and H2O2 since 4 days of incubation. At Day 7 of incubation, the lowest inhibited percentages were observed with NaCl- and H2O2-supplemented plates. While the inhibition percentage was from Days 1–3, NaOH and (NH3)2CO3 plates were completely inhibited, which indicated that green fungus did not generate spores for 3 days after inoculation. However, after 3 days, the inhibition percentages of NaOH and (NH3)2CO3 plates gradually decreased to 75.44 and 52.83%, respectively at Day 7. Ammonium carbonate was found to be effective in reducing the radial growth and completely inhibited the colony growth of A. flavus. As demonstrated by Shekhar et al. (2009), A. flavus that contaminated post-harvest maize is highly sensitive to (NH3)2CO3, which reduced its radial growth at a concentration of 20 mM. Samapundo et al. (2007) found that 1% of (NH3)2CO3 completely inhibited the growth of Aspergillus isolates, which resulted in a large reduction of aflatoxin B1-production in corn. From these results, agar plate supplemented with NaOH presented the highest fungal growth inhibition as confirmed by the fungal hyphal growth at Day 7 in Figure 3c. The diameter of fungal mycelium on NaOH plate was shorter than that of the other chemical plates. The control plate (without chemical in Figure 3a) and the plates supplemented with Na2SO3, NaCl, (NH4)2CO3, KOH, Ca(OH)2 and H2O2 plates, as shown in Figures 3b, 3d and 3e–h, respectively, found that fungi grew and hyphae expanded from the original plug which then distributed over the agar plates.
Figure 2. Fungal growth inhibition percentages of each agar plate supplemented with chemical.
Figure 3. Fungal growth on agar plate supplemented with 10%w/v chemical (A) control (without chemical) (B) Na2SO3 (C) NaOH (D) NaCl (E) (NH4)2CO3 (F) KOH (G) Ca(OH)2 and (H) H2O2.
In case of hole-plate diffusion for seven chemicals selection, the clear zones appeared around test holes of NaOH and H2O2 addition as shown in Figure 4. The appearance of clear zone around the test hole indicated that the fungi could not produce the reproductive spore and could not consequently grow in these areas with presence of chemical solutions. Thus, the aflatoxigenic fungal spore formations were inhibited by addition of NaOH and H2O2 on hole-plate diffusion. A. flavus growth is inhibited by H2O2 as per the study by Kure et al. (2020), and hydrogen peroxide is effective in reducing the spores of A. flavus isolated from food waste.
Figure 4. Fungal growth inhibition by hole-plate method.
The results for seven chemicals selection through disc diffusion are shown in Figure 5; fungi could produce mycelium with fully bloom on sterilized water disc (control disc), and no clear zones were observed around the discs of (NH4)2CO3, Ca(OH)2, Na2SO3 and NaCl. But the discs of NaOH, KOH and H2O2 revealed the clear zone. Fountain et al. (2015) reported that the growths of A. flavus and A. parasiticus were inhibited at a high H2O2 concentration. Therefore, in case of disc diffusion method, it was found that NaOH, KOH and H2O2 inhibited the aflatoxigenic fungal growth.
Figure 5. Fungal growth inhibition by disc diffusion method.
From the seven chemicals selected through three methods of agar plating as shown above, NaOH was the best chemical for aflatoxigenic fungal growth inhibition. The direct exposure of pathogenic fungi to NaOH could destruct the internal structures of fungi or even loss their normal shape after block the metabolic functions of fungal cells. Moreover, A. flavus could not exhibit the conidia production in the strong alkalinity inform of hydroxyl ion such as in NaOH and KOH media (Al-Janabi, 2011; Monzur et al., 2016). Therefore, NaOH was selected for aflatoxin B1 elimination in low-grade maize by heat and chemical treatments.
In this experiment, aflatoxigenic fungi and aflatoxin B1 in low-grade maize were eliminated by treatment with heat and selected chemical (NaOH) from the results of A. flavus growth inhibition in agar plates. The treatment samples were added with NaOH solution at difference concentrations 0, 2.5 and 5%(w/v) and kept at three differential temperatures 25, 50 and 75°C for 24, 48 and 72 h. The suitable conditions for aflatoxigenic fungi and aflatoxin B1 elimination in low-grade maize were evaluated through moisture content, number of aflatoxigenic fungi and aflatoxin B1 content.
The initial moisture content of low-grade maize was 5.31 ± 0.25%, which was increased to 18.09 ± 1.58% after inoculation with 106 spores/mL fungal spores and 25.28 ± 1.15% after addition of NaOH solution.
The effect of temperature on moisture and changes in moisture content during heat and chemical treatments are shown in Figure 6. The moisture contents were significantly different, which were clearly divided into three levels when treated with differential temperatures of 25, 50 and 75°C. The highest moisture content level was observed in the range of 25.57 to 27.81% when treated at 25°C, which was the minimum temperature provided. In low-grade maize samples that were heated at 50°C, the moisture content decreased from 11.34–20.00% and 1.20–8.15% at 75°C. The lowest moisture content level was observed at 75°C, and moreover the moisture content at 75°C decreased faster than that at 50°C. Under conditions of 0% w/v NaOH for 24 h, the moisture contents at 25, 50 and 75°C were observed as 25.57, 22.00 and 8.15%, respectively. Moreover, moisture contents decreased at high temperature (50 and 75°C), under conditions of 0% w/v NaOH; their levels when kept for 24, 48 and 72 h were 22.00, 16.66 and 11.34%, respectively. Therefore, it was observed that high temperature significantly decreased the moisture content levels, which tended to decrease even more when the time of heat was extended. The heat provided to low-grade maize caused the moisture content to decrease rapidly from the initial value to the lowest level. According to the report of Srikiatden and Robert (2007), when the grain receives heat energy during the drying process, the water in grains continuously evaporates by moving to the surface and into the atmosphere. The surface of grains become too dry, and the moisture content gradually decreases to the lowest level at zero drying rate.
Figure 6. Moisture content of heat- and NaOH-treated low-grade maize.
Analysis of variance (ANOVA) and response surface of individual effect and two-factor interactions of temperature, NaOH concentration and time on moisture content is shown in Table 1 and Figure 7. Temperature (A) and time (C) had individual effects on moisture content in low-grade maize at P < 0.05. The two-factor interaction of temperature × time (AC) also had a significant influence on the moisture content at P < 0.05. The increase in temperature and time resulted in the decrease of moisture content in the response surface (Figure 7b). Schultz (2016) reported that fungal growth and reproductive fungal spores could be prevented by keeping the moisture content lower than 14% (w/w) or by lowering the water activity. As shown in Figure 7b, a moisture content level lower than 14 % (w/w) was maintained by high levels of temperature (A) and time (C), which were maintained at higher than 60°C and 30 h.
Table 1. ANOVA of individual effect and two-factor interactions on moisture content.
|P; Prob > F|
|< 0.0001*||< 0.0001*||0.8123||< 0.0001*||0.4983||0.0038*||0.4605|
A = temperature B = NaOH concentration C = time
AB = temperature × concentration AC = temperature × time BC = concentration × time
*Significant level, P < 0.05 R2 = 0.9741 adjust R2 = 0.9663
Figure 7. Response surfaces two factors interaction model of moisture content. (A) Factors interaction of temperature × concentration (AB); (B) temperature × time (AC); (C) concentration × time (BC).
The effect of NaOH concentration on moisture content is presented in Figure 6. NaOH solution with differential concentration 0, 2.5 and 5% w/v had no change on the moisture content. The moisture content levels of low-grade maize samples that were treated at 25°C for 24 h with NaOH concentrations of 0, 2.5 and 5% w/v were 25.57, 26.63 and 25.89%, respectively; and at 50°C for 24 h with NaOH concentrations of 0, 2.5 and 5% w/v were 22.00, 20.00 and 19.84%, respectively. Thus, NaOH concentrations without temperature, had no significant effect on the moisture content (P < 0.05) as shown in ANOVA (Table 1) and response surface (Figure 7.). The interactions between temperature and NaOH concentration (AB) and NaOH concentration and time (BC) did not have any affect on the moisture content (P < 0.05) as shown in Figures 7a and 7c, respectively. Our results found that NaOH concentration did not have any profound effects on the moisture content of low-grade maize. However, the study by Shi et al. (2008) reported that NaOH pretreatment at high temperature resulted in slight diffusion of the moisture; the moisture diffusion coefficient of the surface showed a trend to increase when NaOH treatment was used.
The initial fungal number of aflatoxigenic fungi contaminated in low-grade maize was 2 × 104 cfu/g spores, which changed upon heat and chemical treatments as presented in Figure 8. Aflatoxigenic fungi were able to grow and produce more mycelial spores during treatment at 25°C, and the highest fungal number (8.2 × 104 cfu/g) was observed at 25°C with 0% w/v NaOH for 72 h. The moisture contents at 25°C were in the range of 25.89–27.44%. The moisture content of low-grade maize was higher than 14% which resulted in fungal contamination and production of mycotoxins. It was reported that fungal growth and mycotoxin production in grains could be prevented by maintaining the maximum moisture content level below 14% (Vallverdú et al., 2022). At various temperatures, when low-grade maize samples were treated with 0% w/v of NaOH for 24 h at 25, 50 and 75°C, the fungal numbers were 4.7 × 103, 2.2 × 103 and 0 cfu/g, respectively. The fungal number tended to decrease when the temperature was increased, which confirmed the effect of temperature (A) on fungal number (P < 0.05), as presented in Table 2. Fungi in low-grade maize samples treated at high temperatures (50 and 75°C) found to produce less number of spores. These results were consistent with the findings of Rajarajan et al. (2021), who reported that fungi favoured to grow in temperature ranging from 12–48°C. A. flavus can grow at 37°C and produce the highest levels of mycotoxins at 28°C. Furthermore, heat treatment at 50°C with 0% w/v NaOH for 24, 48 and 72 h found that the number of fungi increased to 2.2 × 103, 3.9 × 103 and 7.3 × 103 cfu/g, respectively. At 50°C without NaOH, fungal spores were observed in low-grade maize because low heat treatment could not destroy the fungal spores. However, fungal spore formation was not observed on addition of NaOH at temperatures 50 and 75°C. Most fungi have an optimal growth rate within a comfortable temperature, and when the temperature reaches a certain point, the growth stops and cell components begin to damage as a result of the heat (Kamil et al., 2011). The interaction term of temperature × time (AC) also had a significant influence on fungal contamination in low-grade maize at P < 0.05, as presented in Table 2. The fungal number tended to decrease when the factors of temperature and time were increased as presented in the response surface (Figure 9b). Through the heat–chemical treatment process, the fungal numbers might increase or decrease based on the conditions of each factor, which affected the induction or inhibition of fungal sporulation.
Table 2. ANOVA of individual effect and two-factor interactions on fungal number.
|P; Prob > F|
A = temperature B = NaOH concentration C = time
AB = temperature × concentration AC = temperature × time BC = concentration × time
*Significant level, P < 0.05 R2 = 0.7315 adjust R2 = 0.6509
Fungal numbers from low-grade maize after treatment with various NaOH concentrations are shown in Figure 8. At 25°C for 24 h with 0, 2.5 and 5% w/v NaOH, fungal numbers were 4.7 × 103, 3.6 × 103 and 3.2 × 103 cfu/g, respectively. The number of fungi after treatment with 2.5 and 5% w/v of NaOH decreased from 0% w/v NaOH. Due to this, NaOH and other alkali are proved to mitigate the aflatoxigenic fungal growth and aflatoxin production (Kumar et al., 2021). According to the finding of Lefyedi and Taylor (2006), the reduction in microbial counts of the green malts is directly related to the NaOH concentration up to 0.2%, because most fungi do not survive at higher pH values. Furthermore, at higher temperature (50°C), the fungal numbers significantly decreased when NaOH concentration was increased, as observed in the treatment of 0, 2.5 and 5% w/v NaOH for 72 h that resulted in 2.2 × 103, 0 and 0 cfu/g, respectively. From Table 2, it can be observed that NaOH concentration was the individual effect on fungal number that led to a significance in the fungal formation (P < 0.05).
Figure 8. Fungal number of heat- and NaOH-treated low-grade maize.
Figure 9. Response surfaces two-factor interaction model of fungal number. (A) Factors interaction of temperature × concentration (AB); (B) temperature × time (AC); (C) concentration × time (BC).
In addition, the two-factor interactions were evaluated by ANOVA to verify their significances on fungal number as presented in Table 2 and respond surface of interaction effect presented in Figure 9 (a–c). The interaction effect of NaOH concentration × time (BC) did not significantly affect the fungal numbers, but the interactions of temperature × NaOH concentration (AB) and temperature × time (AC) significantly influenced the fungal numbers and contamination in low-grade maize at P < 0.05. The interaction between two factors was observed by comparing the two edges of the surface that was not parallel. In case of interaction between temperature (A) and NaOH concentration (B), the high temperature (A) influenced the decrease of fungal number in a more pronounced way when NaOH concentration (B) was increased (Figure 9b). Thus, the co-effects of high temperature and NaOH concentration were effective to reduce the contamination of fungi by destruction of fungal spores, and fungi did not multiply after this treatment. Thermal and alkaline treatments can increase the efficacy of reduction in mycotoxin contamination by mitigating the aflatoxigenic fungal growth (Karlovsky et al., 2016; Kumar et al., 2021). The shape of the response surface suggested that a displacement towards higher concentrations of NaOH and temperature when treatment time was extended led to less number of fungal spore contamination. Within this experimental domain, the lowest predicted number of fungal spore contaminate was 0 cfu/g or fungal spore contamination not found, which was achieved using the following conditions: NaOH concentrations of 2.5 and 5% w/v at 50 and 75°C, with a treatment time of more than 24 h, which corresponded to the conditions in Figure 8. Figure 9 shows that fungal number was not found in low-grade maize in the conditions of tempeature higher than 60°C and heating time higher than 30 h with addition of at least of 2.5% w/v of NaOH.
The number of living fungal spores correlated with the moisture content or water activity in low-grade maize; the decrease in moisture content levels resulted in the reduction of fungal spore number as presented in Figure 10. Low-grade maize containing a moisture content level above 11% was contaminated by fungal spores. Thus, moisture is one of the factors that promote fungal growth. These results were similar to the findings of Genkawa et al. (2008), who reported that fungi were observed in rice with more than 14.4% moisture content at 25°C, however no forms of fungi were observed on rice with less than 12.8% moisture content. Furthermore, the lowest limit for growth of A. flavus happened at moisture ranging from 18 to 25% (Hassane et al., 2017).
Figure 10. Moisture content and fungal number.
Some conditions of heat and NaOH treatment were selected to analyse aflatoxin B1 levels (Table 3). The initial aflatoxin B1 content in low-grade maize before treatment with NaOH was 10.72 µg/kg. After treatment with 2.5 and 5% w/v NaOH at 50 and 75°C, AFB1 decreased. The lowest AFB1 level was found to be 4.25 µg/kg with 60.35% reduction after treatment with 5% w/v NaOH at 50°C for 24 h. Low-grade maize treated with 2.5% w/v NaOH at 75°C for 24 h also showed reduction in aflatoxin B1 content (55.69%).
Table 3. Moisture content, fungal number and AFB1 content in low-grade maize.
|NaOH (% w/v)||Temperature (°C)||Time (h)||Moisture (% db)||Fungal spores (cfu/g)||AFB1 (µg/kg)||% reduction of AFB1|
|1. Low-grade maize without fungi addition||2.68||-||7.52||-|
|2. Low-grade maize with fungi addition||25.62||20,000||10.72||-|
|3. Fungi treatment low-grade maize|
Increasing temperatures (25, 50 and 75°C) with 0% w/v NaOH, moisture contents and numbers of fungi in low-grade maize decreased, and AFB1 was also decreased from 10.72 µg/kg to lower values. The moisture content in low-grade maize was reduced by about 20% from the initial value to 1.80–8.15% w/w at 75°C for 24–72 h. These moisture content levels were not suitable for fungal growth and therefore no contamination with aflatoxigenic fungi was observed. Our findings were in agreement with that of Hassane et al. (2017) who noticed a correlative effect of moisture on fungal growth and aflatoxins production in wheat flour. The growth of A. flavus happen at moisture contents ranging from 18 to 25% and temperature range of 7.5–40°C with optimal growth at around 25°C. These conditions, A. flavus favours to germination and thereby production of aflatoxin. In a study by Lahouar et al. (2016) A. flavus that produced aflatoxin B1 isolated from sorghum seeds, most favoured growth temperature was 25–37°C and water activity 0.91 aw.
Interestingly, after treatment with NaOH at conditions 2.5 and 5% w/v, fungal spores and aflatoxin B1 were absent, and aflatoxin B1 also decreased to a lower than initial value in low-grade maize (7.52 µg/kg). An extension of time on NaOH-treated low-grade maize resulted in decreased amount of aflatoxin B1. Because the molecule of aflatoxin B1 was breaken by NaOH during treatment. This result correlated with the previous research findings that alkaline treatment leads to the opening of the lactone ring of aflatoxin and its transformation to another form called the beta-keto acid compound, a less toxic aflatoxin D1 (Jalili, 2016). Therefore, aflatoxin contamination in low-grade maize was eliminated by heat and NaOH treatment. Heat helps decrease the moisture content, which results in fungus being unable to produce spores and therefore aflatoxin B1 is not produced. NaOH could inhibit the reproductive spores and destruct mycelial spores of aflatoxigenic fungi thereby decreasing AFB1 (Sheikh and Awad, 2022). Furthermore, the study by Memdes et al. (2013) found that thermal alkaline treatment reduced 98% of aflatoxin contamination during roasting process at 250°C for 15 min by adding 20–30 g/kg NaOH in cocoa liquor products. Most aflatoxins are chemically and thermally stable with temperatures up to 100°C, and therefore these factors have little effect on mycotoxin contamination reduction. However, the thermal and chemical treatments increase the efficacy of mitigation of aflatoxin contamination (Karlovsky et al., 2016).
In this study, the aflatoxigenic fungi isolated from low-grade maize with high frequency of occurrence was identified as A. flavus. The growth of A. flavus was used for selection of the chemical, which could inhibit its spore formation; the seven chemicals used were NaOH, (NH4)2CO3, Ca(OH)2, Na2SO3, KOH, NaCl and H2O2. Sodium hydroxide had the potential to inhibit the growth of A. flavus through three agar plate methods. Thus, NaOH was applied as the antifungal growth and aflatoxin B1 detoxifying agent in low-grade maize by heat–-chemical treatment. The contaminated aflatoxigenic fungal low-grade maize was sprinkled with NaOH solution. The treatments were done by varying three factors in three levels of NaOH concentration (0, 2.5 and 5% w/v), temperature (25, 50 and 75°C) and time of exposure (24, 48 and 72 h). Aflatoxin B1 levels were decreased to 17.54% on treatment with 5% w/v NaOH at 25°C (at room temperature) for 48 h, while the treatment using heat at 75°C for 72 h without NaOH addition resulted in the reduction of aflatoxin B1 to 23.97%. However, the highest reduction of aflatoxin B1 (60.35%) was presented in the treatment condition of 5% w/v NaOH at 50°C for 24 h, in which the aflatoxin B1 content decreased to 4.25 µg/kg. Thus, it was observed that treatment using both heat and chemical was better. Heat treatment along with NaOH addition provided more efficiency and significantly reduced the level of fungal contamination, and consequently eliminated the aflatoxin B1 in low-grade maize.
This work was financially supported by the Research and Researcher for Industry (RRi), Thailand Research Fund (TRF) and Phatananikom Kaset Ltd., Company (Thailand) (Grant No. PHD59I0015), and partially supported by Chiang Mai University.
Afzal, H., Shazad, S. and Nisa, S.Q.U., 2013. Morphological identification of Aspergillus species from the soil of Larkana district (Sindh, Pakistan). Asian Journal of Agriculture and Biology 1(3): 105–117.
Aiko, V. and Mehta A., 2015. Occurrence, detection and detoxification of mycotoxins. Journal of Bioscience 40(5): 943–954. 10.1007/s12038-015-9569-6
Al-Janabi, A.A.H.S., 2011. Direct effect of alkaline compounds with hydroxide ions on the viability of dermatophytes. Asian Journal of Pharmaceutical and Biological Research 1: 419–425.
Ali, M., Tumbeh, A.L.S., Izhar, M., Farghal, M., Khattak, A.M., Jah, I., et al., 2020. Melatonin mitigates the infection of Collectotrichum gloeosporioides via modulation of the chitinase gene and antioxidant activity in Capsicum annuum L. Antioxidants 10(7): 1–24. 10.3390/antiox10010007
AOAC Official methods of analysis, 2000. AOAC official methods 925.10, 65.17, 974.24, 992.16. The Association of Official Analytical Chemist, Gaithersburg, MD, USA.
AOAC, Official methods of analysis, 2002. AOAC official method 991.31, Aflatoxins in corn, raw peanuts, and peanut butter. In: Latimer, G.W. (ed.) Official methods of analysis of AOAC International. pp. 21–23. The Association of Official Analytical Chemist, Gaithersburg, MD, USA.
Barnett, H.L. and Hunter, B.B., 1987. Illustrated Genera of Imperfact Fungi, 4th ed. Macmillan, New York, USA, pp. 92–107.
Boonma, S., Rangsee, W. and Chaiklangmuang, S., 2018. Effect of hydrothermal pre-treatment on ferulic acid content and antioxidant activities of corn hydrolysate. Japan Journal of Food Engineering 19(1): 27–34.
Eslami, M., Mashak, Z., Heshmati, A., Shokrzadeh, M. and Nejad, A.S.M., 2015. Determination of aflatoxin B1 levels in Iranian rice by ELISA method. Toxin Reviews 34: 1–4. 10.13140/RG.2.1.3900.4245
Fountain, J.C., Scully, B.T., Chen, Z.Y., Gold, S.E., Glenn, A.E., Abbas, H.K., et al., 2015. Effect of hydrogen peroxide on different toxigenic and atoxigenic isolates of Aspergillus flavus. Toxins 7: 2985–2999. 10.3390/toxins7082985
Gaziano, R., Campione, E., Iacovelli, F., Marino, D., Pica, F., Francesco, P.D., et al., 2018. Antifungal activity of Cardiospermum halicacabum L. (Sapindaceae) against Trichophyton rubrum occurs through molecular interaction with fungal Hsp90. Drug Design, Development and Therapy 12: 2185–2193. 10.2147/DDDT.S155610
Genkawa, A.T.T., Uchino, B.A., Inoue, A.F., Tanaka, B. and Hamanaka, D., 2008. Development of a low-moisture content storage system for brown rice: storability at decreased moisture contents. Biosystems Engineering 4(99): 515–522. 10.1016/j.biosystemseng.2007.12.011
Hassane, A.M.A., El-Shanawany, A.A., Ado-Dahab, N.F., Abdel-Hadi, A. M., Abdul-Raouf, U. M. and Mwanxa, M., 2017. Influence of different moisture contents and temperature on growth and production of aflatoxin B1 by a toxigenic Aspergillus flavus isolate in wheat flour. Journal of Ecology of Health & Environment 5(3): 77–83. 10.18576/jehe/050302
Heshmati, A., Nejad, A.S.M. and Mehri, F., 2021. Occurrence, dietary exposure, and risk assessment of aflatoxins in wheat flour from Iran. International Journal of Environmental Analytical Chemistry, 1–14. 10.1080/03067319.2021.2011254
Jaibangyang, S., Nasanit, R. and Limtong, S., 2021. Effect of temperature and relative humidity on aflatoxin B1 reduction in corn grains and antagonistic activities against aflatoxin-producing Aspergillus flavus by a volatile organic compound-producing yeast, Kwoniella heveanensis DMKU-CE82. BioControl 66: 433–443. 10.1007/s10526-021-10082-x
Jalili, M., 2016. A review on aflatoxins reduction in food. Iranian Journal of Health and Environment 3(1): 445–459.
Kamil, O.H., Lupuliasa, D., Dranganescu, D. and Vlaia, L., 2011. Interrelations of drying heat and different fungal spores within the tablets formulation. Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii 21(2): 339–342.
Karlovsky, P., Suman, M., Berthiller, F., Meester, J.D., Eisenbrand, G., Perrin, I., et al., 2016. Impact of food processing and detoxification treatments on mycotoxin contamination. Mycotoxin Research 32: 179–205. 10.1007/s12550-016-0257-7
Khan, R., Ghazali, F.M., Mahyudin, N.A. and Samsudin, N.I.P., 2020. Mophological characterization and determination of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolated from sweet corn kernels and soil in Malaysia. Agriculture 450(10): 1–13. 10.3390/agriculture10100450
Kumar, A., Pathak, H., Bhadauria, S. and Sudan, J., 2021. Aflatoxin contamination in food crops: causes, detection, and management: a review. Food Production, Processing and Nutrition 3(17): 1–9. 10.1186/s43014-021-00064-y
Kure, C.F., Langsrud, S. and Moretro, T., 2020. Efficient reduction of food related mould spores on surfaces by hydrogen peroxide mist. Foods. 10(55): 1–8. 10.3390/foods10010055
Lahouar, A., Marin, S., Crespo-Sempere, A., Said, S. and Sanchis, V., 2016. Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds. Revista Argentina de Microbiología 48(1): 78–85. 10.1016/j.ram.2015.10.001
Lefyedi, M.L. and Taylor, J.R.N., 2006. Effect of dilute alkaline steeping on the microbial contamination, toxicity and diastatic power of sorghum malt. Journal of the Institute of Brewing 112(2): 108–116. 10.1002/j.2050-0416.2006.tb00240.x
Makhlouf, J., Campos, C.A., Querin, A., Tadrist, S., Puel, O., Lorber, S., et al., 2019. Morphologic, molecular and metabolic characterization of Aspergillus section Flavi spices marketed in Lebanon. Scientific Reports 9: 1–11. 10.1038/s41598-019-41704-1
Memdes, T.A., Scott, P.M. and Tague, B., 2013. Analysis of cocoa products for ocharatoxin A and aflatoxins. Mycotoxin Research 29: 193–201. 10.1007/s12550-013-0167-x
Monzur, M.A., Fakruddin, Md., Hossain, Md. N., Khandaker, R.M. and Abhijit, C., 2016. Growth response of Aspergillus flavus IMS 1103 isolated from poultry feed. Asian Journal of Medical and Biological Research 2(2): 221–228. 10.3329/ajmbr.v2i2.29064
Nejad, B.S., Rajabi, M., Mamoudabadi, A.Z. and Zarrin, M., 2014a. In vitro anti-Candida activity of hydroalcoholic extracts of Heracleum persicum fruit against phatogenic Candida species. Jundishapur Journal of Microbiology 7(1): 1–4. 10.5812/jjm.8703
Nejad, A.S.M., Ghannad, M.S. and Kamkar, A., 2014b. Determination of aflatoxin B1 levels in Iranian and Indian spices by ELISA method. Toxin Reviews 33(4): 151–154. 10.3109/15569543.2014.942319
Okayo, R.O., Andika, D.O., Dida, M.M., K´Otuto, G.O. and Gichimu, B.M., 2020. Morphological and molecular characterization of toxigenic Aspergillus flavus from groundnut kernels in Kenya. International Journal of Microbiology 2020: 8854718. 10.1155/2020/8854718
Pankaj, S.K., Shi, H. and Keener, K.M., 2018. A review of novel physical and chemical decontamination technologies for aflatoxin in food. Trends in Food Science & Technology. 71: 73–83. 10.1016/j.tifs.2017.11.007
Peng, W.X., Marchal, J.LM. and Poel, A.F.B.v.d., 2018. Strategies to prevent and reduce mycotoxins for compound feed manufacturing. Animal Feed Science and Technology 237: 129–153. 10.1016/j.anifeedsci.2018.01.017
Rajarajan, P., Sylvia, K., Periasamy, M.P. and Subramanian, M., 2021. Detection of aflatoxin producing Aspergillus flavus from animal feed in Karnataka, India. Environmental Analysis Health and Toxicology 36(3): 1–9. 10.5620/eaht.2021017
Rushing, B.R. and Selim, M.I., 2019. Aflatoxin B1: a review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food and Chemical Toxicology 124: 81–100. 10.1016/j.fct.2018.11.047
Samapundo, S., Devlieghere, F., Meulenaer, B.D., Lamboni, Y., Osei-Nimoh D. and Debevere J.M., 2007. Interaction of water activity and bicarbonate salts in the inhibition of growth and mycotoxin production by Fusarium and Aspergillus species. International Journal of Food Microbiology 116: 266–274. 10.1016/j.ijfoodmicro.2007.01.005
Sanders, E.R., 2012. Aseptic laboratory techniques: plating methods. Journal of Visualized Experiments 63: 1–18. 10.3791/3064
Schultz, C., 2016. Water activity as related to microorganisms in the manufacturing environment. General Internal Medicine and Clinical Innovations 1(6): 1–2. 10.15761/GIMCI.1000133
Sheikh, H. and Awad, M.F., 2022. Biogenesis of nanoparticles with inhibitory effects on aflatoxin B1 production by Aspergillus-flavus. Electronic Journal of Biotechnology 60: 26–35. 10.1016/j.ejbt.2022.09.003
Shekhar, M., Singh, S., Khan, A.A.A. and Kumar, S., 2009. Efficacy of Inorganic salts and organic acids against colony growth of Aspergillus flavus and their use to control aflatoxin level in post-harvest maize. Journal of Food Safety 11: 4–10.
Shi, J., Pan, Z., Mchugh, T.H., Wood, D., Zhu, Y., Avena-Bustillos, R.J., et al., 2008. Effect of berry size and sodium hydroxide pretreatment on the drying characteristics of blueberries under infrared radiation heating. Journal of Food Science 73(6): 259–265. 10.1111/j.1750-3841.2008.00816.x
Sipos, P., Peles, F., Brassó, D.L., Béri, B., Pusztahelyi, T., Pócsi, I., et al., 2021. Physical and chemical methods for reduction in aflatoxin content of feed and food. Toxins 13(204): 1–17. 10.3390/toxins13030204
Srikiatden, J. and Robert, J.S., 2007. Moisture transfer in solid food materials: a review of mechanisms, models, and measurements. International Journal of Food Properties 10: 739–777. 10.1080/10942910601161672
Thanaboripat, D., Nontabenjawan, K., Leesin, K., Teerapiannont, D., Sukcharoen, O. and Ruangrattanamatec, R., 1997. Inhibitory effect of garlic, clove and carrot on growth of Aspergillus flavus and aflatoxin production. Journal of Forestry Research 8 (1): 39–42. 10.1007/BF02864939
Thathana, M.G., Murage, H., Abia, A.L.K. and Pillay, M., 2017. Morphological characterization and determination of aflatoxin-production potentials of Aspergillus flavus isolated from maize and soil in Kenya. Agriculture 70(8): 1–14. 10.3390/agriculture7100080
Tian, F. and Chun, H.S., 2017. Natural product for preventing and controlling aflatoxin contamination of food. In: Abdulra’uf, L. (ed). Aflatoxin—control, analysis, detection and health risks. IntechOpen, London, UK, pp. 13–44.
Vallverdú, B.B., Ramos, A.J., Martínez, C.C., Marín, S., Sanchis, V. and Ortega, J.F., 2022. Influence of agronomic factors on mycotoxin contamination in maize and changes during a 10-day harvest-till-drying simulation period: a different perspective. Toxins. 14 (620): 1–15. 10.3390/toxins14090620