Introduction
Aflatoxins are toxic secondary metabolites produced by fungal species from the genus Aspergillus, especially A. flavus, A. parasiticus and A. nomius (de Oliveira et al., 2014; Wochner et al., 2018). Although more than 20 types of aflatoxins were identified, aflatoxin B1 (AFB1) is considered the major toxic metabolite produced by the fungi contaminating foods and feeds. The International Agency for Research on Cancer has classified AFB1 as group 1 carcinogens (European Food Safety Authority [EFSA], 2020). Considering the toxic effects of AFB1, proper prevention and control strategies in the feed and food chains are of fundamental importance to avoid health issues (Schrenk et al., 2020). Prevention of aflatoxin contamination of foodstuffs can be achieved through the following two main approaches: inhibiting the fungal growth and subsequent aflatoxin biosynthesis, and/or detoxifying toxic compounds already produced by the fungi in food matrices (Ismail et al., 2018). Strategies for inhibiting fungal growth include employing improved agricultural practices, antifungal agents, genetic engineering, and appropriate storage conditions (Corassin et al., 2013). Decontamination of aflatoxins could be achieved by using physical, chemical and biological tools aimed at inactivating or degrading toxic substances in food matrices (Agriopoulou et al., 2020). Physical techniques, such as heat-based inactivation, irradiation and ultrasound (US) application on food, have shown promising results for inactivating or degrading aflatoxins (Grenier et al., 2014).
Wheat is a vital food ingredient consumed globally and utilized extensively in a wide range of food preparations. For example, wheat, because of its high nutritional values, is usually employed to produce daily-based breads, cookies, cakes and other essential edible products (Grenier et al., 2014). Fermented wheat-based products are prepared by using two vital substances: starch, a carbohydrate polymer that serves as a significant energy source, and gluten, a protein predominantly found in wheat that imparts elasticity and softness to dough (Ma et al., 2022; Xiong et al., 2021). Consequently, developing effective technologies for decontamination of wheat containing aflatoxins has gained considerable interest in scientific and economic communities. Previous works investigating the effect of fermentation on the levels of aflatoxins in fermented wheat doughs indicated that this process could reduce levels of aflatoxin in final products (Noroozi et al., 2020, 2022; Rashid Hudu et al., 2022).
Among emerging technologies for processing of food, US has emerged as an innovative and promising method for different applications in the food industry (Abedi et al., 2019; Dumuta et al., 2022; Gavahian et al., 2020). As the name suggests, ultrasound refers to inaudible sound waves with frequencies higher than those detectable by the human ear (20 kHz). Regarding its applications, high-frequency (>1 MHz) and low-intensity (<1 W cm2) US, and low-frequency (20–100 kHz) with high-intensity (10–1,000 W cm2) US have demonstrated notable uses in the food industry (Alarcon-Rojo et al., 2015). The application of high-intensity US in food processing has been considered advantageous because of its operation at lower temperature with minimal impact on the physicochemical properties of foods (Nunes et al., 2021). This technology has the potential to inactivate spoiling microbes and pathogenic enzymes in food products and beverages (Dumuta et al., 2022). Moreover, US is an eco-friendly and nonpolluting technique. Given the significant effect of US on aflatoxin degradation (Liu et al., 2019b), possible interactions between fermentation and US detoxification have been hypothesized (Gavahian et al., 2021). US can degrade AFB1 by affecting the chemically stable furan moiety and by changing lactone ring in the main structure of AFB1 (Gavahian et al., 2021; Liu et al., 2019b). Therefore, the aim of this study was to conduct a preliminary investigation to evaluate the effect of US, alone or in combination with fermentation, on the level of AFB1 in wheat flour-based doughs.
Material and methods
Ingredients, reagents and equipment
Wheat flour samples were obtained in the summer of 2022 from local retailers at Pirassununga, São Paulo state, Brazil. Prior to the experiment, the collected wheat flour samples were submitted for aflatoxin analysis as described in subsequent sections, and were free of aflatoxins. The AFB1 standard (Sigma-Aldrich, St. Louis, MO) was dissolved in acetonitrile and diluted to achieve a working solution containing 1.0-μg AFB1/mL. This solution was used for spiking wheat flour samples with AFB1 at 50 ng/g of AFB1 for preparing contaminated doughs used in decontamination assays.
The spiking process was accomplished by transferring appropriate aliquots of AFB1 working solution and wheat flour to a 250-mL beaker. The contents were mixed thoroughly and left in an oven at 50°C for evaporation of solvent. The US equipment (SolidSteel, Piracicaba, SP) used in the study had a frequency of 40 kHz with a power output of 100 W (Guo and Lei, 2020). AFB1 analysis of the samples was conducted using a high-performance liquid chromatography (HPLC) system (Shimadzu®, Kyoto, Japan), equipped with a fluorescence detector (excitation: 360 nm, and emission: 440 nm) and a Kinetex C18 column (Phenomenex, Torrance, CA, USA) with a dimension of 4.6 × 150 mm (2.6 μm), preceded by a Shim-pack pre-column (5 μm, 4 × 10 mm). All reagents used in the experiment were of HPLC grade.
Preparation of wheat dough
Experimental wheat doughs were prepared following the procedures described by Mousavi-Khaneghah et al. (2023) and using the main ingredients as presented in Table 1.
Table 1. Main ingredients for preparing experimental wheat doughs.
| Ingredients | Quantity |
|---|---|
| Wheat flour (spiked with aflatoxin B1 [AFB1]) | 100.0 g |
| Water | 50.0 mL |
| Dry yeast (Saccharomyces cerevisiae) | 1.0 g |
| Vegetable fat | 14.0 g |
| Salt | 0.8 g |
| Sodium bicarbonate | 1.5 g |
| Total | 167.3 |
All ingredients (AFB1-contaminated wheat flour, sodium bicarbonate, vegetable fat, salt, and dry yeast) were mixed in a glass container. The experimental doughs were prepared in duplicate lots; one lot was submitted directly for US treatments, and the other lot was reserved for fermentation prior to subjecting to US treatments. The fermentation of experimental doughs was conducted at 28°C for 18 h in a fermentation cabinet with 85% relative humidity (RH). In addition, fermented and non-fermented control doughs (without AFB1) were prepared and subjected to the above-described US treatments.
Total titratable acidity (TTA) was evaluated as an indicator of fermentation process, using the method described by Li et al. (2013). TTA was measured in triplicate according to the method described by García-Mantrana et al. (2015) using 10-g dough fermented at 28°C for 2 h (relative humidity 85%) and mixed with 100 mL of acetone–water solution (5:95 ratio, v/v) for 5 min., and titrated subsequently. The results were expressed in volume (mL) of 0.1-N NaOH to titrate 10 g of fermented dough (García-Mantrana et al., 2015).
Ultrasound treatment
Aflatoxin B1 decontamination assays using the experimental doughs subjected to US treatments were performed as described by Liu et al. (2019b) with small modifications. Briefly, a 25-g aliquot of non-fermented and fermented doughs was weighed in Falcon tubes and introduced into a US bath set at 40 kHz, 100 W/cm2 and 35°C for 10, 30 and 60 min. The tubes were removed from the US bath at the end of each treatment period and the samples were immediately submitted for AFB1 determination.
Analysis of aflatoxin B1
Aflatoxin B1 in the samples was determined following the method recommended by manufacturer of the immunoaffinity columns (Aflatest®; Vicam, Milford, MA), followed by HPLC with fluorescence detector. The entire content of the sample in the Falcon tube (25 g) was mixed with 5 g of NaCl and 125 mL of methanol–water solution (70:30 ratio, v/v). After stirring the content in a shaker for 30 min, 15 mL of extract was centrifuged at 2,000 ×g and filtered through a filter paper into a beaker. Following this step, 30 mL of the ultra-purified water (Milli-Q; Millipore, Burlington, MA) was added to the extract, after which the mixture was homogenized and filtered again through a 1.5-μm filter membrane (Millipore, Burlington, MA). The collected 15-mL extract was passed through immunoaffinity column, connected to a vacuum system (flow set at 1–2 drops/s). After sample elution, the column was washed with 10-mL Milli-Q water. Then, 1 mL of HPLC-grade methanol was passed through eluted AFB1, collecting the eluate in an amber vial. The eluate was evaporated to dryness under N2 flow and derivatized with 100 μL of trifluoroacetic acid and 200 μL of n-hexane (Jager, 1990). The mixture was homogenized and placed in an oven at 35ºC for 10 min., evaporated again, and finally re-suspended in 500 μL of ethanol–water solution (50:50 ratio, v/v). Final extracts were filtered through 0.45-μm polytetrafluoroethylene polymer (PTFE) filtering membranes (Millex; Millipore, Burlington, MA) and presented for chromatographic analysis.
The determination of AFB1 was carried out by injecting a 20 μL sample extract into the HPLC system (Shimadzu 10 VP, Kyoto, Japan) as described above, using water–methanol–acetonitrile solution (60:20:20, v/v) as mobile phase with a flow rate of 1.0 mL/min. Under these conditions, the retention period of AFB1 was approximately 3.8 min. Calibration curves were prepared with AFB1 working solution and derivatized exactly as described previously to reach concentrations of 1.56, 3.125, 6.25, 12.5, and 25.0 ng/mL. The quantification of AFB1 in samples was performed through the interpolation of the areas of chromatographic peaks and the regression equation of calibration curves. The limit of detection (LoD) and limit of quantification (LoQ) were calculated based on signal-to-noise ratios of 3:1 and 10:1, respectively.
Performance of analytical method
To evaluate the accuracy of the analytical method used for determining AFB1 in wheat samples, recovery tests were carried out as described by Jager et al. (2016). Dough samples were prepared as described previously and spiked with AFB1 working solution to achieve contamination levels of 5.0 and 50.0 µg/kg. The fortified samples were prepared in triplicate (n = 3) for each AFB1 level, and subjected to the method described in previous sections to determine recovery proportions and coefficient variances of analytical results.
Statistical analysis
AFB1 levels in the samples from different treatments were submitted to analysis of variance, according to the procedures established in the General Linear Model of SAS® (Vonesh, 2012). Tukey’s test was used for comparison of mean values, when applicable, considering μ = 0.05.
Results and Discussion
The TTA of fermented dough samples subjected to US treatments ranged between 3.0±0 mL and 4.0±0 mL (data not shown), and no differences (P < 0.05) were observed between treatments. These results indicated that the fermentation process, as estimated by TTA (García-Mantrana et al., 2015), was not affected by any US treatment employed in the present experiment. The data presented in this work agreed with the previous studies indicating that US at small frequencies associated with the fermentation resulted in improved enzymatic activity in various food products (Gavahian et al., 2021).
Analytical performance for determining aflatoxin B1 in wheat dough samples
The performance evaluation of the analytical method used in this study is crucial for ensuring reliability of the results. Table 2 presents concentration values, recoveries, and coefficients of variation obtained for the analytical method used for AFB1 quantification in experimental dough samples. Data indicate that accuracy and precision attributes were satisfactory (Azevedo et al., 2021), considering the average recoveries of 85.0% and 88.4% and coefficients of variation of 7.9% and 11.5% achieved for samples spiked with 5.0 ng/g and 50.0 ng/g, respectively.
Table 2. Concentrations, recoveries and coefficients of variance of analytical method for determination of aflatoxin B1 in wheat-based doughs.
| Aflatoxin B1 added (ng/g) | Aflatoxin B1 obtained (ng/g) | Aflatoxin B1 medium (ng/g) | Aflatoxin B1 recovery (%) | Coefficient of variance (%) |
|---|---|---|---|---|
| 0 | 0 0 0 |
0 | - | - |
| 5 | 3.84 4.12 4.79 |
4.25 | 85.0 | 11.5 |
| 50 | 47.40 40.45 44.75 |
44.20 | 88.4 | 7.9 |
The LoD and LOQ were 0.1 ng/g and 0.4 ng/g, respectively. The analytical method proved as effective and sensitive to determine the used concentrations of AFB1 in experimental dough samples. Figure 1 shows typical chromatograms obtained for AFB1 in wheat-based dough samples spiked with 50-ng/g toxin before and after 60 min of US treatment.
Figure 1. Chromatograms showing the retention time of aflatoxin B1 (AFB1) (nearly 3.8 min) in: (A) wheat-based dough spiked with 50 ng/g of AFB1 before ultrasound treatment and fermentation; (B) non-fermented, spiked wheat-based dough after 60 min of US treatment (35.72 ng/g); and (C) fermented and spiked wheat-based dough after 60 min of US treatment (21.40 ng/g).
Ultrasound treatment of the fermented and non-fermented wheat doughs
The presence of AFB1 in foods, including wheat-based products, is a major concern because of its harmful properties (EFSA, 2020; Schrenk et al., 2020). Therefore, implementing suitable methods to reduce AFB1 contamination in food products is an important issue to prevent health problems. Among these methods, US, as investigated in the present work, has emerged as a promising technique because of its easy availability, low cost, and effectiveness in reducing mycotoxins in foods (Dumuta et al., 2022; Nunes et al., 2021).
The concentration and percentage reduction of AFB1 in fermented and non-fermented doughs treated with US at different periods are shown in Table 3. The non-fermented samples subjected to US treatment alone had significant reduction (P < 0.05) in AFB1 levels, which decreased from 50.0 ng/g (nominal value) to 42.24±7.62 ng/g after 10 min, and further to 26.99±9.09 ng/g after 60 min. However, dough samples subjected to the combined methods of US and fermentation exhibited even lower (P < 0.05) levels of AFB1, decreasing from 50.0 ng/g (nominal value) to 23.20±6.42 ng/g after 10 min, and to 19.33±3.78 ng/g after 60 min. Consequently, the percentage reduction of AFB1 levels in non-fermented doughs varied between 3.6% and 46.0% and between 6.7% and 61.7% in fermented doughs (Table 3). Reduction in AFB1 levels because of US treatment alone, as observed in this study, was consistent with findings of previous studies conducted on the application of US for decontamination of mycotoxins (Liu et al., 2019a, 2022).
Table 3. Concentration and percentage reduction of aflatoxin B1 in wheat-based doughs by ultrasound treatment submitted to different periods.
| Type of dough | Applied ultrasound duration (min) | |||||||
|---|---|---|---|---|---|---|---|---|
| 0 | 10 | 30 | 60 | |||||
| ng/g | %1 | ng/g | %1 | ng/g | %1 | ng/g | %1 | |
| Non-fermented | 48.21±3.42A,a | 3.6 | 42.24±7.62A,a | 15.5 | 37.89±1.83A,b | 24.2 | 26.99±9.09A,c | 46.0 |
| Fermented | 46.67±6.16A,a | 6.7 | 23.20±6.42B,b | 53.6 | 21.99±5.58B,b | 56.0 | 19.33±3.78B,b | 61.3 |
A,B,a,b,cDifferent uppercase letters in the columns, or different lowercase letters in the rows differ by the Tukey’s test at P < 0.05.
1Percentage reduction calculated in relation to the nominal concentration of AFB1 before treatments (50.0 ng/g).
Ultrasound is a physical method that generates cavitation, resulting in localized high temperatures and pressures that can disrupt microorganisms (Khadhraoui et al., 2021). These disruptions can also lead to changes in physicochemical properties, thereby enhancing the degradation of mycotoxins (Gavahian et al., 2021; Liu et al., 2019a). In addition, US treatment generates several free radicals, such as hydrogen atoms and hydroxyl, which can degrade AFB1 molecules (Liu et al., 2019a). Hence, reduction in AFB1 levels observed in wheat dough samples treated with US is potentially attributed to a combination of these effects.
Formation of AFB1-degraded products after US treatment was not assessed in the present study. However, Liu et al. (2019b) reported mass spectrometry data, indicating that eight new degradation products were generated after 40 min of US treatment to degrade AFB1. The structures of these eight reaction products resembled that of parent compound, with modifications in furofuran and lactone rings and the methoxy group of AFB1 moiety (Liu et al., 2019b).
Regarding the application of combined treatments of US and fermentation, an additional reduction effect was observed on the levels of AFB1 in wheat dough samples (Table 3), with longer periods of US treatment resulting in greater reductions (P < 0.05). This suggests a possible synergistic interaction between the two methods that could enhance the decontamination efficiency of AFB1 by US in the fermented product. However, the mechanism by which such potential interaction reduces the levels of AFB1 in fermented wheat flour dough samples has to be determined.
The fermentation process lowers the pH of wheat dough (Casado et al., 2017), which further inhibits the growth of microorganisms, such as AFB1-producing fungi, rendering them more susceptible to the effects of US treatment (Khadhraoui et al., 2021). However, the effect of US on microbial cells can vary according to different parameters, such as sonication time, temperature, and pH (Gavahian et al., 2021). These variables could also impact the decontamination effect of US in fermented wheat doughs evaluated in the present study. The higher AFB1 degradation activities observed in the US-treated fermented doughs could also be attributed to the stress exerted by the magnetic field of US and the application of combined fermentation, when compared to traditional approaches used in similar contexts (Gavahian et al., 2021). However, the efficiency of sonication depends on factors such as the chemical structure of mycotoxins and possibly matrix-associated effects, which were not assessed in the present study.
Conclusion
Results of the present study indicate that US treatment, particularly when used in combination with fermentation, is an effective method for reducing AFB1 levels in fermented wheat dough samples. Besides, the US treatment did not interfere with the TTA of fermented doughs. The best combination for decontamination of AFB1 was fermentation associated with US application for 10 min, although the potential effects of temperature and pH on the decontamination process of AFB1 were not assessed in this preliminary investigation. The future studies are required to optimize the process conditions of AFB1 decontamination and determine possible effects of US on the sensory attributes of wheat-based products. Although US treatment reduces AFB1 levels, it must not be considered as a substitute for good agricultural practices and proper storage conditions to prevent fungal growth and mycotoxin contamination.