Occurrence and Exposure Assessment of Mycotoxins from Beers Commercially Traded in Brazil

Abstract

Mycotoxins are toxic secondary metabolites produced by fungi that often contaminate food materials used in beer production, posing health risks to consumers. This study investigated the occurrence and levels of mycotoxins in commercial beers commercially available in São Paulo, Brazil, and assessed the estimated daily intake (EDI) of quantifiable mycotoxins. Sixty beer samples from different brands and compositions (barley malt, malt with corn, and malt with rice) were analyzed for deoxynivalenol (DON); aflatoxins (AFs) B₁, B₂, G₁, and G₂; ochratoxin A (OTA); T-2 toxin; fumonisins (F) (B₁ and B₂); and zearalenone (ZEN) using ultra-performance liquid chromatography coupled to tandem mass spectrometry. FB₁ was quantified in all samples, while DON, ZEN, OTA, AFB₁, and T-2 toxin were detected in 40, 65, 25, 20, and 10%, respectively. Mean levels of 2.38, 36.41, 0.19, 1.05, 0.78, and 0.47 ng/mL were observed for FB₁, DON, ZEN, OTA, AFB₁, and T-2 toxin, respectively. Mycotoxin co-occurrence was observed in 43 (71.7%) samples analyzed, with DON and FB₁ as the most frequent combination (20%). The EDI values of individual mycotoxins were generally below tolerable daily intakes established by international agencies. However, the co-occurrence of up to four different mycotoxins in beers warrants concern on the possible interactive toxic effects of mycotoxin mixtures and reinforces the necessity of specific regulations for ready-to-drink beverages in Brazil.

1. Introduction

Beer is one of the most consumed alcoholic beverages worldwide, standing out in both the Brazilian and global markets. In Brazil, it accounted for 91.9% of the country’s total volume of alcoholic beverage consumed in 2022 [1]. The high consumption of this alcoholic beverage raises concern regarding the possible contamination of raw materials and the final products with natural toxins, such as mycotoxins, the secondary toxic metabolites produced by fungi species in some genera such as Aspergillus, Penicillium, and Fusarium [2,3]. Mycotoxin contamination at different levels in major food ingredients used in beer production has been described, including barley, malt, hops, or their adjuncts [4]. The main mycotoxins found in many agricultural products are the aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisins (FB), and T-2 toxin [5]. These contaminants are frequently studied due to the significant risks they pose to public health, especially regarding their chronic effects such as carcinogenicity, mutagenicity, and immune suppression [6].

The presence of these toxic substances in beers can occur at various stages of production, mainly by transference from cereals to malt and subsequently to beer due to their high thermal stability and water solubility [7]. Therefore, it is crucial to rigorously control these contaminants at all stages, from agricultural production to final food processing [8]. The continuous ingestion of these contaminants can lead to chronic toxic effects, exposing consumers to levels above those considered safe for health [2]. In Brazil, maximum permitted limits (MPLs) for mycotoxins in beers are not available, although regulations for mycotoxins in several types of foods have been established, including MPLs for AF, DON, ZEN, OTA and FBs in food ingredients used for beer production [9]. Research studies have been conducted in various countries to identify the co-occurrence of these substances in beers, highlighting that high beer consumption can represent an important source of dietary mycotoxins [7]. Despite the popularity of beer and the relevance of its safety for public health, limited data are available on the occurrence of mycotoxins in Brazilian commercial beers. In addition, studies on the simultaneous occurrence of multiple mycotoxins in beers available for consumption in Brazil have not been conducted. Therefore, this study aimed to determine multiple mycotoxins in beers commercially traded in the state of São Paulo, Brazil. The estimated daily intake (EDI) of quantifiable mycotoxins was also determined, to assess their dietary exposure through the consumption of Brazilian beers. The findings of this study contribute to understanding the potential exposure to mycotoxins through the consumption of beers in Brazil.

2. Materials and Methods

2.1. Sampling Procedures

All beers evaluated in this study were manufactured and pasteurized by companies located in the state of São Paulo. Sixty beer samples from twenty distinct brands, using different combinations of ingredients and selected based on their popularity in Brazil, were collected from supermarkets in the state of São Paulo, Brazil. Among the 60 samples collected, 30 were made with malt only, 24 were manufactured with malt and corn, and 6 were made with malt and rice. Samples were stored at room temperature until analysis to determine the presence and concentration of DON, AFB₁, AFB₂, AFG₁, AFG₂, OTA, T-2 toxin, FB₁, FB₂, and ZEN.

2.2. Mycotoxin Analysis of Beer Samples

The extraction and clean-up procedures for determination of co-occurring mycotoxins in beer samples were carried out using a commercially available MycoSpin™ 400 multi-mycotoxin column, following the procedures as recommended by the manufacturer (Romer Labs, Getzersdorf, Austria) with some modifications, as follows. Initially, 5 mL of each beer sample was transferred to a Falcon tube and placed in an ultrasonic bath for 5 min to decarbonate until the foam in the sample significantly decreased. Next, 1 mL of the decarbonated beer was pipetted into an Eppendorf tube and centrifuged at 6000× g for 5 min to separate the solids from the liquid. The supernatant (800 µL) was carefully transferred into a new 2 mL Eppendorf tube, and 800 µL of acetonitrile plus 80 µL of acetic acid were introduced to the supernatant, followed by mixing in a vortex shaker. The mixture was then centrifuged again at 6000× g for 5 min. After this process, 750 µL of the liquid was applied into the MycoSpin™ 400 column, and the mixture was vortexed for 1 min. After this step, the bottom of the column was detached according to the manufacturer’s instructions and centrifuged for 1 min at 6000× g. Finally, the supernatant (750 µL) was dried in a MultiVap 64™ concentrator (LabTech, Sorisole, Italy) and re-suspended in 340.9 µL of 10% acetonitrile, followed by 1 min of mixing in a vortex shaker. The resulting liquid was passed through a 0.22 µm PTFE membrane before being transferred to a 2 mL vial. Subsequently, 40 µL of the sample and 10 µL of an internal standard (IS) solution, previously prepared in water/acetonitrile (1:1, v/v) containing [¹³C₁₇]-AFB₁, [¹³C₂₀]-OTA, [¹³C₃₄]-FB₁, [¹³C₁₈]-ZEN, and [¹³C₂₄]–T2 at 100 ng/mL and [¹³C₁₅]–DON at 750 ng/mL, were introduced into an insert placed in another 2 mL vial. The mixture was vortexed and subjected immediately to mycotoxin analysis.

Mycotoxins in beer samples were determined using an Acquity I-Class™ (Waters Corp., Milford, MA, USA) ultra-performance liquid chromatography (UPLC) equipped with a BEH Column C18 (2.1 × 50 mm, 1.7 µm) and coupled to tandem mass spectrometry (MS/MS) Xevo TQS™ equipment (Waters, Milford, MA, USA). The mobile phase was composed by water (eluent A) and acetonitrile (eluent B), both containing 0.1% of formic acid. A gradient flow was used for elution of injected samples, eluent A kept at 95% for 0.5 min, followed by linear increase in eluent B to 25% over 4.5 min. After this period, eluent B was increased to 90% over 0.5 min, followed by a hold time of 0.25 min. After that, percentage of eluent B was reduced to 5% over 0.5 min, and the column re-equilibrated to the initial conditions for 0.5 min. The total chromatographic run time was 6.5 min, and the mobile phase flow rate was kept at 0.5 mL/min. The MS/MS was operated in Multiple Reaction Monitoring (MRM) mode, using ionization by electron pulverization with positive and negative ion modes. The operating conditions were as follows: capillary voltage: 0.75 kV; source temperature: 150 °C; desolvation temperature: 500 °C; desolvation gas flow: 800 L h⁻¹; cone gas flow: 150 L h⁻¹. Cone voltage, collision energy, and MRM transitions (major precursor ion > fragment ion) were manually optimized for individual mycotoxins [10]. The MRM quantification, confirmation transitions for mycotoxins, and optimized mass spectrometer parameters used are presented in Appendix A,

Table A1. Analytical parameters of the method for determination of mycotoxins in beer samples.

Mycotoxin	Mass (g/mol)	Molecular Ion	Transition (m/z)
AFB1	312.3	[M + H]+	312.7 > 284.9 a
			312.7 > 241.1 b
[13C17]–AFB1	329.1	[M + H]+	330.3 > 301.5
AFB2	314.3	[M + H]+	314.7 > 259.0 a
			314.7 > 287.0 b
AFG1	328.3	[M + H]+	328.9 > 243.0 a
			328.9 > 199.5 b
AFG2	330.3	[M + H]+	330.9 > 245.0 a
			330.9 > 188.9 b
OTA	403.1	[M + H]+	404.0 > 238.9 a
			404.0 > 357.9 b
[13C20]–OTA	423.7	[M + H]+	424.2 > 250.0
FB1	721.8	[M + H]+	722.5 > 334.0 a
			722.5 > 352.1 b
[13C34]–FB1	755.6	[M + H]+	756.6 > 374.4
FB2	705.8	[M + H]+	706.5 > 336.2 a
			706.5 > 318.3 b
DON	296.3	[M + H]+	297.3 > 249.1 a
			297.3 > 231.1 b
[13C15]–DON	311.2	[M + H]+	312.1 > 98.7
ZEN	318.1	[M − H]−	317.1 > 175.1 a
			317.1 > 130.9 b
[13C18]–ZEN	336.2	[M − H]−	335.1 >185.1
T2	466.2	[M + NH4]+	484.2 > 305.2 a
			484.2 > 185.0 b
[13C24]–T2	490.3	[M + NH4]+	508.3 > 322.1

^a Transitions used for quantification; ^b Transitions used for confirmation. AFB₁: aflatoxin B₁; AFB₂: aflatoxin B₂; AFG₁: aflatoxin G₁; AFG₂: aflatoxin G₂; OTA: ochratoxin A; FB₁: fumonisin B₁; FB₂: fumonisin B₂; DON: deoxynivalenol; ZEN: zearalenone; T2: T-2 toxin.

. Calibration curves were constructed with five concentrations of each mycotoxin. A graph of peak area vs. concentration was plotted, and the regression value (r) was calculated. A residual plot, representing the difference between the true value (measured signal) and the predicted value from the equation of the line for each analyte concentration, was also prepared to visually assess the goodness of fit of the regression model of calibration curves. Data collection and processing were carried out in MassLynx software version 4.1.

2.3. Estimation of Probable Daily Intake of Mycotoxins

By using the results obtained in the analyses, the EDI of each mycotoxin quantified in samples was calculated based on Equation (1) [11,12].

$$\mathrm{E}\mathrm{D}\mathrm{I}={\displaystyle \frac{\mathrm{b}\mathrm{e}\mathrm{e}\mathrm{r} \mathrm{i}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\left(\mathrm{m}\mathrm{L}/\mathrm{d}\mathrm{a}\mathrm{y}\right)\times \mathrm{m}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} (\mathrm{\mu }\mathrm{g}/\mathrm{m}\mathrm{L})}{\mathrm{b}\mathrm{o}\mathrm{d}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{k}\mathrm{g}\right)}}$$

(1)

The EDI values were estimated using the lowest and highest intakes as the minimum and maximum, respectively, for each mycotoxin detected and quantified in beer samples [6]. An average body mass of 72 kg for men and 60 kg for women was used, based on data for the Brazilian population [13]. Additionally, an average beer consumption of 50.5 L per year, or 0.14 L per day was adopted [14].

2.4. Performance of the Analytical Method

The performance of the analytical method was evaluated using a pool of beer samples previously analyzed and found to have non-detectable levels of mycotoxins. The limits of detection (LOD) for each mycotoxin were as follows: DON = 2.04 ng/mL; AFB₁ = 0.08 ng/mL; AFB₂ = 0.09 ng/mL; AFG₁ = 0.09 ng/mL; AFG₂ = 0.17 ng/mL; OTA = 0.06 ng/mL; T2 = 0.11 ng/mL; FB₁ = 0.16 ng/mL; FB₂ = 0.18 ng/mL; ZEN = 0.03 ng/mL. Ten mL of this blank beer pool were transferred into five Falcon tubes and spiked with aliquots of a mycotoxin mixture standard solution, to achieve 5 concentrations for each mycotoxin in each tube, as follows: Tube 1: 0.25 ng/mL of AFB₁, AFB₂, AFG₁, AFG₂, OTA, and ZEN + 3.0 ng/mL of FB₁ and FB₂ + 1.5 ng/mL of T2 + 12.0 ng/mL of DON; Tube 2: 0.5 ng/mL of AFB₁, AFB₂, AFG₁, AFG₂, OTA, and ZEN + 6.0 ng/mL of FB₁ and FB₂ + 3.0 ng/mL of T2 + 15.0 ng/mL of DON; Tube 3: 1.0 ng/mL of AFB₁, AFB₂, AFG₁, AFG₂, OTA, and ZEN + 9.0 ng/mL of FB₁ and FB₂ + 6.0 ng/mL of T2 + 20.0 ng/mL of DON; Tube 4: 2.0 ng/mL of AFB₁, AFB₂, AFG₁, AFG₂, OTA, and ZEN + 12.0 ng/mL of FB₁ and FB₂ + 10.0 ng/mL of T2 + 30.0 ng/mL of DON; Tube 5: 4.0 ng/mL of AFB₁, AFB₂, AFG₁, AFG₂, OTA, and ZEN + 22.0 ng/mL of FB₁ and FB₂ + 20.0 ng/mL of T2 + 50.0 ng/mL of DON. The number of repetitions was three for each mycotoxin level, totaling 15 Falcon tubes containing the prepared spiked beer samples. These samples were analyzed exactly as described in Section 2.2, to determine recovery rates and repeatability relative standard deviations (RSDr) [15]. The LOD and the limit of quantification (LOQ) were determined by reducing the analyte concentration to a value where the signal obtained was three and 10 times the baseline noise, respectively.

3. Results

3.1. Performance of Analytical Method

Table 1. Performance of the analytical method for determination of mycotoxins in beer samples.

Mycotoxin	Spiking Level a (ng/mL)	RT (min)	Mean Recovery (%)	RSD (%)	LOD (ng/mL)	LOQ (ng/mL)
AFB1	0.25–4.0	4.99	87.1	19.1	0.08	0.26
AFB2	0.25–4.0	4.69	85.9	16.8	0.09	0.28
AFG1	0.25–4.0	4.66	90.7	27.8	0.09	0.31
AFG2	0.25–4.0	4.67	81.8	17.5	0.17	0.56
OTA	0.25–4.0	5.71	99.0	12.5	0.06	0.21
FB1	3.0–22.0	5.41	102	15.3	0.16	0.52
FB2	3.0–22.0	5.77	110	21.9	0.18	0.59
DON	12.0–50.0	2.12	95.3	19.2	2.04	6.80
ZEN	0.25–4.0	6.03	98.7	26.6	0.03	0.10
T2	1.5–20.0	5.97	79.1	8.10	0.11	0.37

^a n = 3 for each spiking level. RT: retention time; RSD: relative standard deviation; LOD: limit of detection; LOQ: limit of quantification; AFB₁: aflatoxin B₁; AFB₂: aflatoxin B₂; AFG₁: aflatoxin G₁; AFG₂: aflatoxin G₂; OTA: ochratoxin A; FB₁: fumonisin B₁; FB₂: fumonisin B₂; DON: deoxynivalenol; ZEN: zearalenone; T2: T-2 toxin.

presents the performance of analytical method for determination of mycotoxins in beer samples. The overall values obtained for the mean recovery ranged from 79.1 to 110%, while the RSD values varied between 8.1 and 27.8%. The LOD and LOD values ranged from 0.03 to 2.04 and 0.10 to 6.80 ng/mL, respectively.

3.2. Occurrence of Mycotoxins in Beer Samples

Table 2. The occurrence of mycotoxins in beers sold in the state of São Paulo, made from barley malt, malt and corn, and malt and rice.

	Main Ingredients of Beers
	Malt (n = 30)		Malt and Corn (n = 24)		Malt and Rice (n = 6)		Total (n = 60)
Mycotoxin	n (%)	Mean (Range) (ng/mL)	n (%)	Mean (Range) (ng/mL)	n (%)	Mean (Range) (ng/mL)	n (%)	Mean (Range) (ng/L)
AFB1	5 (16.7)	0.70 (0.37–0.94)	5 (20.8)	0.77 (0.35–1.16)	2 (33.3)	0.87 (0.76–0.97)	12 (20)	0.78 (0.35–1.16)
AFB2	1 (3.3)	0.80	0	<LOQ	0	<LOQ	0	<LOQ
AFG1	0	<LOQ	0	<LOQ	0	<LOQ	0	<LOQ
AFG2	0	<LOQ	0	<LOQ	0	<LOQ	0	<LOQ
OTA	7 (23.3)	1.16 (1.08–1.30)	6 (25)	0.93 (1.03–1.12)	2 (33.3)	1.06 (1.05–1.07)	15 (25)	1.05 (1.03–1.30)
FB1	30 (100)	2.15 (1.23–4.41)	24 (100)	2.44 (1.21–4.75)	6 (100)	2.02 (1.32–2.99)	60 (100)	2.38 (1.23–20.58)
FB2	5 (16.7)	2.33 (1.70–4.81)	5 (20.8)	3.68 (1.71–4.97)	1 (16.7)	1.71	11 (18.3)	2.57 (1.70–4.97)
DON	10 (33.3)	37.22 (12.79–86.47)	11 (45.8)	19.99 (10.25–55.62)	3 (50)	26.17 (19.90–31.72)	24 (40)	36.41 (10.25–86.47)
ZEN	18 (60)	0.19 (0.16–0.28)	17 (70,8)	0.20 (0.16–0.29)	4 (66.7)	0.17 (0.17–0.18)	39 (65)	0.19 (0.16–0.29)
T2	5 (16.7)	0.59 (0.52–0.83)	1 (4.2)	0.83	0	<LOQ	6 (10)	0.47 (0.52–0.83)

n: number of samples containing levels higher than the limit of quantification (LOQ). See Table 1 for LOQ of each mycotoxin. AFB₁: aflatoxin B₁; AFB₂: aflatoxin B₂; AFG₁: aflatoxin G₁; AFG₂: aflatoxin G₂; OTA: ochratoxin A; FB₁: fumonisin B₁; FB₂: fumonisin B₂; DON: deoxynivalenol; ZEN: zearalenone; T2: T-2 toxin.

presents the occurrence levels of mycotoxins in beer samples, according to the main ingredients used in the production process. The frequency and levels of mycotoxins in positive samples varied among different ingredients, thus reflecting the importance of the contamination status of the food ingredients used in beer production. AFB₁ was found in 20% of the samples, with a mean concentration of 0.78 ng/mL (range: 0.35–1.16 ng/mL). AFB₁ was more common in beers made from malt and rice, with a detection rate of 33.3%. However, AFG₁ and AFG₂ were below the LOQ. OTA was present in 25% of the samples, with a mean concentration of 1.05 ng/mL (range: 1.03–1.30 ng/mL). The highest average concentration of OTA was observed in beers made from pure malt (1.16 ng/mL), although the highest detection frequency was observed in beers made from malt and rice, with 33.3%. Therefore, beers with pure malt had a higher concentration of OTA, while those with malt and rice had a higher incidence.

FB₁ was detected in all samples (100%), with a mean concentration of 2.38 ng/mL (range: 1.23–20.58 ng/mL), while FB₂ was present in 18.3% of samples, with a mean of 2.57 ng/mL (range: 1.70–4.97 ng/mL). Beers made from malt and corn had the highest average concentration of FB₁ (2.44 ng/mL). ZEN was also more frequent in beers made from malt and corn, with a detection rate of 70.8%, with a mean concentration of 0.19 ng/mL (range: 0.16–0.29 ng/mL). Therefore, the use of corn contributed to a higher incidence of FB₁ and ZEN. These results suggest the need for continuous monitoring and possible revisions in production and storage processes to reduce mycotoxin contamination in beers. The widespread presence of FB₁ may be related to the use of contaminated corn or other grains in beer production.

DON was detected in 40% of the total samples analyzed, with a mean concentration of 36.41 ng/mL (range: 10.25–86.47 ng/mL). Beers made from pure malt showed a detection frequency of 33.3% and the highest average concentration of DON (37.22 ng/mL). Beers combining malt and corn had a higher incidence (45.8%), but a lower average concentration (19.99 ng/mL). Beers made from malt and rice showed the highest incidence (50%), with an intermediate concentration of 26.17 ng/mL. Therefore, DON was more frequent in beers with malt and rice, but with a higher average concentration in beers made from pure malt. The highest average concentration of AFB₁ was found in beers made from malt and rice (0.87 ng/mL), followed by those made from malt and corn (0.77 ng/mL) and pure malt (0.70 ng/mL). T-2 toxin was detected in 10% of samples, with a mean concentration of 0.47 ng/L (range: 0.52–0.83 ng/mL).

Table 3. Mycotoxin co-occurrence in beer samples sold in the state of São Paulo, Brazil.

Types of Co-Occurring Mycotoxins	Number of Samples Positive for Mycotoxins (n = 60) a
DON, FB1	12
AFB1, FB1	6
DON, OTA, FB1	5
OTA, FB1	4
DON, AFB1, FB1	4
T2, FB1	4
AFB1, OTA, FB1	4
OTA, T2, FB1	2
AFB1, AFB2, FB1	1
DON, AFB1, OTA, FB1	1
Total (%)	43 (71.7)

^a Samples containing levels higher than the limit of quantification (LOQ). See Table 1 for LOQ of each mycotoxin. DON: deoxynivalenol; FB₁: fumonisin B₁; OTA: ochratoxin A; AFB₁: aflatoxin B₁; AFB₂: aflatoxin B₂; T2: T-2 toxin.

presents the mycotoxin co-occurrence in the analyzed beers, with 71.7% of samples presenting two or more mycotoxins. The most frequent combination was DON and FB₁, present in 20% of the samples. The widespread presence of FB₁ across all samples, coupled with its frequent co-occurrence with DON, AFB₁, and OTA, indicates a possible general contamination problem in the ingredients used for beer production. The high co-occurrence of DON and FB₁ also suggests a shared source of contamination, perhaps from grains contaminated in the field.

3.3. Estimated Daily Intake

Assessing the exposure of mycotoxins through beer consumption is essential to ensure the safety of this beverage for the population.

Table 4. Estimated daily intake of mycotoxins from beer consumption by the Brazilian population.

Mycotoxin	Estimated Daily Intake (μg/kg Body Weigh/Day)
	Men (Min.–Max.)	Women (Min.–Max.)
AFB1	0.0007–0.0023	0.0008–0.0027
AFB2	0.0015–0.0016	0.0018–0.0019
OTA	0.0020–0.0025	0.0024–0.0030
FB1	0.0029–0.0050	0.0034–0.0059
FB2	0.0033–0.0100	0.0040–0.0120
DON	0.0199–0.1680	0.0239–0.2019
ZEN	0.0003–0.0006	0.0004–0.0007
T2	0.0010–0.0016	0.0012–0.0019

AFB₁: aflatoxin B₁; AFB₂: aflatoxin B₂; OTA: ochratoxin A; FB₁: fumonisin B₁; FB₂: fumonisin B₂; DON: deoxynivalenol; ZEN: zearalenone; T2: T-2 toxin.

presents the EDI values for each quantified mycotoxins through the consumption of beers.

The EDI for DON presented the highest values for both men (0.0199 µg/kg body weight (bw)/day) and women (0.0239 µg/kg bw/day), followed by FB₂. In contrast, the maximum EDI values were considerably higher, with DON again exhibiting the highest values (0.168 µg/kg bw/day for men and 0.2019 µg/kg bw/day for women), indicating greater exposure in scenarios of high consumption or increased contamination. In general, women presented slightly higher EDI values than men, possibly due to the relatively lower women’s mean bw.

4. Discussion

The results on the performance evaluation of the analytical method indicated that satisfactory recovery and RSD values were obtained [15], thus enabling its application for the simultaneous determination of mycotoxins in beer samples. The highest RSD values were observed for ZEN (26.6%) and AFG₁ (27.8%), which may be associated with variations in the adsorption ability of these compounds by MycoSpin™ 400 columns during clean-up procedures of beer samples. However, all the obtained RSD values were within the acceptable values according to the Eurachem Guide [15] for compounds analyzed in complex matrices at parts per billion (ppb) levels.

The occurrence data presented in this study demonstrate the presence of multiple mycotoxins in commercial beer samples, with a notable prevalence of FB₁, detected in 100% of the analyzed samples. This high prevalence may be attributed to the frequent use of corn as an adjunct in beer production in the state of São Paulo, in addition to the region’s climatic conditions that may favor the proliferation of Fusarium spp. A previous study conducted in Brazil found FB₁ in 49% of beer samples [16], which is lower than the 100% incidence observed in the present study. Compared with our results, a South African study also reported a lower frequency of beer samples containing FB₁ (53%) [17]. In contrast, FB₂ was detected in 32% of the samples, a higher percentage than the 18.3% found in this study. These variations suggest regional differences in mycotoxin contamination, potentially influenced by environmental factors and local agricultural practices. DON has also been frequently reported in beers from various countries. In studies conducted in China and Poland, DON was detected in 93.7 and 96.0% of beer samples, respectively, indicating a widespread occurrence in those regions [18,19]. Similar studies in Germany and Paraguay reported prevalence rates of 75 and 24.1%, respectively [20,21]. In contrast, the present study revealed a substantially lower incidence than those reported in several countries, with DON detected in 40% of the analyzed samples. These findings suggest regional variation in DON contamination, likely linked to differences in food materials, storage conditions, and brewing practices. The lower incidence observed in our samples may also reflect reduced contamination of wheat or its derivatives, which are common DON carriers. Moreover, DON’s high water solubility facilitates its transfer into beer during brewing [18].

The detection of T-2 toxin in 10% of the analyzed samples is a relevant finding, since there is no MPL established in Brazil for this compound in food ingredients or in beers. The absence of specific regulations highlights the need for regulatory updates to mitigate potential health risks to consumers. ZEN was identified in 65% of the analyzed samples, a percentage similar to that reported in other studies, which found incidences varying from 53 to 65% [18,19]. Regarding AFB₁, the percentage of positive samples observed in this investigation was 20%, which is higher than the values reported in China, where 12.5% of samples contained this toxin [18]. AFB₁ is considered the most toxic AF, and this difference may be associated with variations in storage methods and processing of raw materials used in beer production. The presence of AFs, even in small concentrations, warrants concern because they are known to cause cancer. OTA was identified in 25% of the analyzed samples. This percentage is higher than that reported in beers commercialized in Portugal, where the occurrence was 10.6% [22], but significantly lower than that observed in Polish beer samples, in which the mycotoxin was detected in 93% of the cases [19]. These differences may reflect regional variations in the ingredients used, climatic conditions, storage methods, or beer production processes. These findings highlight the need for careful monitoring and effective strategies to reduce mycotoxin contamination throughout the beer production process, from selecting raw materials to storing and processing them.

Considering that co-occurrence of DON and FB₁ was observed in 20% of the beer samples analyzed in this study, the potential combined toxic effects of these mycotoxins deserve attention. Both DON and FB₁ are commonly found together in grains because they affect similar crops and are favored by the same environmental conditions [23,24]. Studies have shown that co-exposure to these toxins can intensify toxic effects, compared with exposure to each one individually. For instance, in vitro studies with porcine kidney cells (PK-15) demonstrated reduced antioxidant enzyme activity and increased oxidative stress when both toxins were present [25]. Mice exposed simultaneously to DON and FB₁ developed pronounced intestinal inflammation, villous atrophy, and impaired digestion, confirming the potential for synergistic effects in vivo [26]. Co-exposure to DON and FB₁ in human intestinal cells (Caco-2) intensified toxic effects, resulting in increased production of reactive oxygen species (ROS), inhibition of protein synthesis, and DNA damage, including methylation and fragmentation [27]. A later in vivo study in mice showed that the combination of these mycotoxins increased DNA methylation in kidney cells, caused irregularities in renal filtration function, and elevated serum creatine kinase levels, indicating tissue necrosis [28]. Interestingly, in human immune cells (THP-1 line), DON and FB₁ together sometimes showed reduced cytotoxicity compared to each toxin alone, potentially due to decreased activation of stress-related signaling pathways [29]. These findings suggest that the combined toxicity of DON and FB₁ may vary depending on the cell type and exposure conditions, reinforcing the importance of considering co-exposure scenarios in food safety risk assessments for products such as beer.

A comparison of the obtained results with the MPLs established by Brazilian regulation [9], Chinese National Food Safety Standard [30], the Codex Alimentarius general standard for contaminants [31], and the European Union regulation (EU) 2023/915 [32] demonstrated that all the mycotoxins detected were at concentrations well below the MPLs established for food ingredients used for beer production. Also, the EDI values obtained for mycotoxins through beer consumption in the present study were below the tolerable daily intake (TDI) reference values for OTA (0.016 μg/kg bw/day) [33], FB₁ plus FB₂ (2.0 μg/kg bw/day) [34], DON (1.0 μg/kg bw/day) [35], and ZEN (0.25 μg/kg bw/day [36]. However, the co-occurrence of up to four different mycotoxins in beers warrants concern on the possible interactive toxic effects of mycotoxin mixtures and reinforce the necessity of specific regulations for ready-to-drink beverages in Brazil.

While the analytical approach used in this study demonstrated high sensitivity and selectivity, some limitations must be acknowledged. The study analyzed a limited number of commercial brands purchased from markets in the state of São Paulo, which may not represent the entire market. Mycotoxin occurrence can vary due to seasonal factors and production-related parameters, which were not controlled in the experimental design used in this study due to logistical and economic constraints. Further research is needed to identify the exact sources of contamination and evaluate the potential health risks associated with the presence of these mycotoxins in beer. Future studies should also be conducted focusing on beers produced in other Brazilian states, also extending the analytical method to include Alternaria toxins such as tenuazonic acid and alternariol, which have been detected in beers and are gaining attention as emerging mycotoxins [37,38]. Given the scarcity of data on the occurrence of these compounds in beers marketed in Brazil, investigating their presence could contribute significantly to food safety assessments and regulatory discussions in the national context.

5. Conclusions

The results of this trial demonstrate the frequent co-occurrence of mycotoxins in beers produced in Brazil, although the quantified levels were below the established regulatory limits for food ingredients used in the beer manufacture. Despite the low EDI values observed for individual mycotoxins, the occurrence of multiple mycotoxins in a sample reinforces the need for continuous monitoring of the raw materials used in beer production. As the current Brazilian legislation for foodstuffs provides MPLs for AFs, FBs, OTA, ZEN, and DON, the inclusion of regulations for other mycotoxins such as T-2 toxin should be considered, as this compound was detected in some samples in this study. In addition, the improvement of preventive measures in the production chain, such as good agricultural practices, proper storage, and specific regulations for ready-to-drink beverages, are required to ensure their safety consumption by the Brazilian population.