Incidence and Levels of Aflatoxin M1 in Artisanal and Manufactured Cheese in Pernambuco State, Brazil

Cheese is one of the most susceptible dairy foods to accumulating aflatoxins due to their high affinity to caseins. The consumption of cheese contaminated with high levels of aflatoxin M1 (AFM1) can be highly harmful to humans. The present work, based on high-performance liquid chromatography (HPLC), highlights the frequency and levels of AFM1 in coalho and mozzarella cheese samples (n = 28) from the main cheese-processing plants in Araripe Sertão and Agreste in the state of Pernambuco, Brazil. Of the evaluated cheeses, 14 samples were artisanal cheeses and the remaining 14 were industrial (manufactured) cheeses. All samples (100%) had detectable levels of AFM1, with concentrations ranging from 0.026 to 0.132 µg/kg. Higher levels (p < 0.05) of AFM1 were observed in artisanal mozzarella cheeses, but none of the cheese samples exceed the maximum permissible limits (MPLs) of 2.5 µg/kg established for AFM1 in cheese in Brazil and 0.25 µg/kg in the European countries by the European Union (EU). The high incidence of low levels of AFM1 found in the evaluated cheeses underscores the need for stringent control measures to prevent this mycotoxin in milk used for cheese production in the study area, with the aim of protecting public health and reducing significant economic losses for producers.


Introduction
Aflatoxins are mycotoxins produced as secondary metabolites by species of the fungal genus Aspergillus, mainly A. flavus, A. parasiticus, and A. nomius, during growth on food and feed products [1]. These fungi produce a range of toxic metabolites, but the main compounds produced under natural conditions are aflatoxins B 1 (AFB 1 ), G 1 (AFG 1 ), B 2 (AFB 2 ), and G 2 (AFG 2 ) [2,3]. All the aflatoxins are highly toxic to humans, causing various effects such as hepatotoxicity, mutagenicity, teratogenicity, immunosuppression, and carcinogenicity, among other effects [1][2][3]. When animals ingest feed contaminated with the most prominent and highest carcinogenic AFB 1 , the compound is biochemically converted in the animal's liver into the hydroxylated aflatoxin M 1 (AFM 1 ), which is excreted in the

Results
This study is the first to evaluate the incidence and levels of AFM 1 in different cheeses from the main production plants in the Araripe Sertão and Agreste regions of Pernambuco state in Brazil. The occurrence and levels of AFM 1 were determined by HPLC, as given in Table 1. The results display a high rate of AFM 1 occurrence in all analyzed cheese samples (100%, n = 28). In addition, the levels of AFM 1 were assessed, with artisanal mozzarella cheese (25%, n = 7) demonstrating levels ranging from 0.026 µg/kg to 0.093 µg/kg, and the manufactured mozzarella cheese (25%, n = 7) indicating levels ranging from 0.037 to 0.132 µg/kg. Similarly, artisanal coalho cheese (25%, n = 7) exhibited AFM 1 levels ranging from 0.035 µg/kg to 0.045 µg/kg, whereas the manufactured coalho cheese (25%, n = 7) revealed AFM 1 levels ranging from 0.035 µg/kg to 0.046 µg/kg. Throughout the analyzed cheese samples, the mean level of AFM 1 for 25% of the artisanal mozzarella cheeses (n = 7) was 0.07 ± 0.02 µg/kg, which was significantly higher (p < 0.05) than the remaining artisanal or manufactured coalho cheeses. To support the results, the levels of AFM 1 in cheese samples were further assessed by distributing them into quartiles ( Figure 1). In this analysis, the statistical comparison displayed that artisanal mozzarella cheese (AM) had a low concentration range of 0.026 µg/kg. An increased variability was found in the AFM 1 levels, which could be compared with the remaining analyzed cheeses. Overall, AFM 1 values for AM ranged from 0.0423 µg/kg to 0.0889 µg/kg, but some samples had concentrations below or above 0.0262 µg/kg and 0.0935 µg/kg, respectively. The median AFM 1 concentration in AM was 0.068 µg/kg.
To support the results, the levels of AFM1 in cheese samples were further assessed by distributing them into quartiles ( Figure 1). In this analysis, the statistical comparison displayed that artisanal mozzarella cheese (AM) had a low concentration range of 0.026 µg/kg. An increased variability was found in the AFM1 levels, which could be compared with the remaining analyzed cheeses. Overall, AFM1 values for AM ranged from 0.0423 µg/kg to 0.0889 µg/kg, but some samples had concentrations below or above 0.0262 µg/kg and 0.0935 µg/kg, respectively. The median AFM1 concentration in AM was 0.068 µg/kg. However, the manufactured mozzarella (MM) cheese revealed AFM1 values ranging from 0.037 µg/kg, which is the minimum limit, to 0.132 µg/kg, which is the maximum limit. Furthermore, 75% of the MM samples had concentrations above 0.0373 µg/kg, and the median concentration was lower compared to the other groups, although there was overlap in quartile 1 with the lower limit.
The analysis of the artisanal coalho (AC) cheese represented AFM1 levels at a minimum of 0.035 µg/kg and a maximum of 0.045 µg/kg, with a median of 0.04 µg/kg. In turn, the manufactured cheese (MC) revealed AFM1 levels ranging from 0.035 µg/kg to 0.047 µg/kg, with a median of 0.041 µg/kg. However, the MC had greater variability in the data compared to the AC ( Figure 1).

Discussion
This study, based on HPLC, evaluated the incidence and levels of AFM1 in cheeses collected from production plants in Araripe Sertão and Agreste in the state of Pernambuco However, the manufactured mozzarella (MM) cheese revealed AFM 1 values ranging from 0.037 µg/kg, which is the minimum limit, to 0.132 µg/kg, which is the maximum limit. Furthermore, 75% of the MM samples had concentrations above 0.0373 µg/kg, and the median concentration was lower compared to the other groups, although there was overlap in quartile 1 with the lower limit.
The analysis of the artisanal coalho (AC) cheese represented AFM 1 levels at a minimum of 0.035 µg/kg and a maximum of 0.045 µg/kg, with a median of 0.04 µg/kg. In turn, the manufactured cheese (MC) revealed AFM 1 levels ranging from 0.035 µg/kg to 0.047 µg/kg, with a median of 0.041 µg/kg. However, the MC had greater variability in the data compared to the AC ( Figure 1).

Discussion
This study, based on HPLC, evaluated the incidence and levels of AFM 1 in cheeses collected from production plants in Araripe Sertão and Agreste in the state of Pernambuco in Brazil ( Table 1). The results demonstrated a high occurrence rate of AFM 1 in all analyzed cheese samples (100%, n = 28) with varying levels ( Table 1). None of the analyzed cheeses had AFM 1 levels higher than the Brazilian MPL of 2.5 µg/kg [20] or the EU MPL of 0.25 µg/kg. In exception to the northeastern region, such as Pernambuco, the AFM 1 evaluation has been shown to be present in a large number of studies conducted in various regions and states of Brazil, as described. This study, for the first time, aimed to evaluate the occurrence and levels of AFM 1 in different types of cheeses produced in the northeastern region of Brazil. These results indicate that consuming cheeses in these regions may be potentially harmful, and proper care, evaluation, and strict quality control procedures are necessary. This is because fungi are toxic organisms that naturally cause contamination of foods and feeds. When present, these toxigenic fungi produce mycotoxins, such as AFB 1 and its hydroxylated AFM 1 , in foods. The hydroxylated AFM 1 can transfer from one food (milk) to another food (e.g., cheeses) during fabrication, even under strict processing and handling procedures. When the resulting cheese products contain high levels of AFM 1 , Toxins 2023, 15, 182 4 of 10 they can pose a risk to human health. To reduce this risk exposure and increase awareness in the food industry, the occurrence and levels of AFM 1 are evaluated in various milk products, comprising different cheeses produced in Brazil.
Brazil is a major producer of cheese in the world, fabricating a wide variety of cheeses. According to the recent data reported by the Food and Agriculture Organization (FAO), around 59,543.14 tons of cheeses were produced in Brazil in 2020 [21]. The country is known for its diverse array of artisanal cheeses, many of which are considered national treasures [22]. Recently, public incentives for technological progress and commercial partnerships for cheese production have increased in Brazil [22]. This has led to growing concerns about the quality and reliability of cheese products. In the last few decades, many studies have been conducted to quantify and evaluate the occurrence level of AFM 1 in different types of cheeses produced in various regions of Brazil. The investigation of AFM 1 in this study is in line with previous data demonstrating the incidence of AFM 1 in different types of cheeses produced not only in Brazil, but also in other countries ( Table 2). Table 2. Incidence and level of aflatoxin M 1 (AFM 1 ) in different types of cheese manufactured globally.
Following additional research, a study conducted in Lebanon (Table 2) found that 67.57% (n = 75/111) of the locally fabricated or imported white and yellow cheese types, comprising Halloumi, Naboulsi, Feta, Baladi, and Akkawi, were contaminated with high occurrence of AFM 1 at levels ranging from 0.00561 to 0.315 µg/kg [31]. However, the same study found that the occurrence and levels of AFM 1 tremendously exceeded the EU permissible level of 0.25 µg/kg [31]. Additionally, data from Iran revealed that 53.8% (n = 194/360) of the locally produced Lighvan, Koozeh, Siahmazgi, Khiki Talesh, and Lactic cheeses were contaminated with higher levels of AFM 1 at 0.0505-0.3087 µg/kg [33]. A study carried out in Mexico City found that 57% (n = 17/30) of Oaxaca cheeses tested contained AFM 1 at an average level of 1.7 µg/kg [34]. In addition, it was found that the Oaxaca artisanal cheeses produced in Veracruz contained higher levels of AFM 1 compared to Oaxaca cheeses fabricated in Mexico City [34]. Additional examination revealed 85% (n = 39/46) of various types of cheeses had higher occurrence of AFM 1 , with a mean level of 0.1977 µg/kg [36]. A report from Ethiopia found 100% (n = 82/82) of the cottage cheese samples tested positive for AFM 1 , with levels recorded at 5.58 ± 0.08 µg/kg [38]. In a recent study, approximately 70% (n = 42/60) of the cheeses produced in Serbia were found to be contaminated with AFM 1 at levels exceeding 0.25 µg/kg [37]. The analysis of white cheese samples (n = 10/25) from Turkey indicated the incidence of AFM 1 at concentrations ranging Toxins 2023, 15, 182 6 of 10 from 0.00246 to 0.035 µg/kg (mean: 0.01714 ± 0.0042 µg/kg) [32]. Similarly, a quantifiable range of AFM 1 was detected in 29% (n = 8) of the Minas frescal cheese manufactured in São Paulo State, at levels ranged from 0.113 to 0.092 µg/kg [12]. However, more studies have been conducted to investigate the presence of AFM 1 in Minas frescal cheese marketed in the northeast region of São Paulo, Brazil [27]. The incidence of this mycotoxin in foods has been shown to be dependent on several factors, including the type of food, seasonal variability, geographic location, post-harvest period, cheese-making procedures, analytical method, and cheese maturation [32,39].
The results of the coalho and mozzarella cheeses can be attributed to variations in manufacturing methods and physicochemical characteristics of each product, as described in previous studies [40,41]. The levels of AFM 1 in cheese depend on the type of cheese, the amount of water eliminated, the curd temperature, the pH of the saturated brine, and the duration of cheese pressing. Reports suggest that AFM 1 levels may be higher in hard cheese compared to soft cheese, due the higher amount of the milk proteins, mainly casein, to which AFM 1 has a greater binding affinity [19]. A study in Brazil evaluated the incidence and levels of AFM 1 in a range of milk and milk-derived products, containing several cheese samples (n = 57) [17]. The study found that of the analyzed products, except a high occurrence rate, only one cheese samples represented AFM 1 at a level of 0.695 µg/kg [17]. Moreover, HPLC coupled with fluorescence-detector-based analyses of cheeses (n = 58), incorporating Minas frescal light, Minas frescal, Minas standard, and other type of cheese, found that 67.24% of the cheeses were contaminated with AFM 1 (0.01-0.304 µg/kg), as given in Table 2 [28].
This work from the northeastern state of Pernambuco in Brazil detected lower levels of AFM 1 compared to other studies conducted on almost same cheese samples in northern Brazil [11]. Similarly, the levels of AFM 1 in artisanal and manufactured mozzarella cheeses ranged from 0.026 to 0.093 µg/kg and from 0.037 to 0.132 µg/kg, respectively, while artisanal and manufactured coalho cheeses showed 0.035-0.045 µg/kg and 0.035-0.046 µg/kg, which were comparatively lower than those detected in northern Brazil. In the northern region, especially the state of Amazonas in Brazil, 25 cheese samples (coalho, coalho de buffalo, mozzarella, mozzarella de buffalo, and Minas frescal) were analyzed, and none of the samples exceeded the Brazilian MPL of 2.5 µg/kg for AFM 1 [11]. Moreover, in the southern region of Brazil, AFM 1 was analyzed in Serrano artisanal cheeses at four different maturation periods (14, 21, 28, and 35 days), and it was observed that only four samples had AFM 1 levels of 0.505, 0.875, 0.093, and 1.03 µg/kg [13]. While most of the samples had AFM 1 levels below the Brazilian MPL of 2.5 µg/kg, some exceeded the EU MPL of 0.25 µg/kg. Although seasonal factors were not considered in the current study, the incidence and level of AFM 1 in cheese samples analyzed can also be influenced by different time periods of cheese manufacture. The artisanal and coalho or mozzarella cheeses produced between March and May 2022 (Brazilian autumn) give AFM 1 levels ranging from 0.026 to 0.132 µg/kg (Table 1).
It is important to note that the impact of seasonality on AFM 1 levels in cheeses has already been described [42]. In this study, the levels of total aflatoxins and AFB 1 in roughage, concentrate, and compound feed were low in the autumn, followed by the summer and winter, while spring had the highest level of mycotoxins [42]. This demonstrates that harvesting and proper drying of vegetable crops used for feed during the autumn may be less risky, leading to milk and cheese with lower AFM 1 levels. Another study found a significant difference (p < 0.05) in the occurrence and level of AFM 1 in traditional cheese produced in the summer and winter seasons [33]. In regard to the seasonal impact on cheese contaminated with AFM 1 , further studies have also been performed. For example, in a study of traditionally produced Egyptian cheeses over two years (2016-2017), the occurrence and concentration of AFM 1 in karish Egyptian cheese was found to range from 1.11 to 0.632 µg/kg [35]. However, a seasonal evaluation exhibited that the karish cheeses were significantly contaminated with AFM 1 at 1.34 µg/kg (2016) and 0.855 µg/kg (2017) only in the winter, compared to the other three seasons [35].
In the current study, none of the analyzed cheese samples exceeded the MPL of 2.5 µg/kg set by Brazil [20]. This limit (2.5 µg/kg) is higher compared to the MPL set by the EU of 0.25 µg/kg for dairy products [9], as well as other countries including Iran, Austria and Switzerland, and Italy, which have set MPLs of 0.25 µg/kg and 0.45 µg/kg [27]. When comparing the results, 25% of the artisanal mozzarella cheese samples showed AFM 1 levels at 0.07 µg/kg that do not exceed any of the described MPLs. Conversely, in a study of parmesan cheese commercially available in Rio de Janeiro, Brazil, overall samples showed AFM 1 levels below the Brazilian MPL of 2.5 µg/kg, but 26.7% samples still exceeded the EU's MPL of 0.25 µg/kg [29]. A study in Ethiopia found that 100% (n = 82) of locally produced and industrialized cheese contained AFM 1 at levels of 5.58 ± 0.08 µg/kg, exceeding the limits set by Egypt, the EU, and Morocco, and 88% of the samples even exceeded the MPL settled by the United States [38].
In a study carried out in Qatar, halloumi and kashkaval cheeses were found to have significantly higher levels of AFM 1 compared to mozzarella, edam, cheddar, cream, and moshalal varieties [36]. The authors attributed the elevated levels of AFM 1 in halloumi and kashkaval cheeses to factors such as maturation time, manufacturing method (industrial or artisanal), milk source, and time of production [36]. The variations in the processing of artisanal cheese were likely due to a lack of standardization, especially in the case of mozzarella cheese, which was observed during the sampling time. Factors contributing to these issues could include the use of poor-quality products, water content, incorrect pH correction, improper cooking, and cooking time of the curdled dough. The production of artisanal cheeses is a traditional method that is passed down from generation to generation and has significant economic importance, especially for small-scale producers. This production is often based on empirical methods, which can result in varying quality standards in the same production region [40,43,44].

Conclusions
According to the findings of this study, a high incidence of AFM 1 was detected using HPLC in both artisanal and industrially produced cheeses in Pernambuco, Brazil. The levels of AFM 1 were low and all the samples were found to be below the limit of 2.5 µg/kg established in Brazil and 0.25 µg/kg adopted in the EU. The study found significant differences between the levels of contamination of artisanal mozzarella-type cheese and manufactured coalho cheese. Based on these results, there is a need for strict quality control measure to further reduce the presence of this mycotoxin in the milk used for cheese manufacture in the studied area. Moreover, future studies should also evaluate the cheeses throughout different seasons to determine the incidence and level of AFM 1 , as seasonality can have a significant impact on cheese production in the same regions of Pernambuco, Brazil.

Sampling Procedures
In this study, a non-probabilistic convenience sampling was employed, which accounted for approximately 70% of the producers in the study area. Participation in the research was voluntary and not all producers agreed to take part. The collection of the samples was performed for the first time in two main cheese-processing plants in the Agreste and Araripe Sertão regions of Pernambuco state, Brazil. A total of 28 samples of mozzarella-and coalho-type cheeses, 14 of which were artisanal and the remaining 14 were industrially produced, were collected between March and May 2022. Individual samples (original package, 500 g) were stored at 4 • C prior to transport to the laboratory for immediate analytical procedures.

Sample Preparation
The analysis of the cheese samples was performed according to a previously in-house validated analytical method [30]. Individual samples (8 g) were taken in falcon tubes Toxins 2023, 15, 182 8 of 10 (15 mL). To each sample was added 2 g of sodium chloride (NaCl), 22 mL of methanol (CH 3 OH, Dinâmica ® , São Paulo, SP, Brazil), and 13 mL of ultra-pure water (Mili-Q). Individual mixtures after homogenization (1 min), shaking (10 min), and centrifugation (6000 rpm × 15 min) were filtered through membrane filters (0.22 µm) in glass tubes. At this stage, 20 mL of the individual extracts were diluted in 30 mL of ultra-pure water (Mili-Q) and submitted to re-centrifugation (6000 rpm × 15 min). Now the final filtrates were subjected to passing through the immunoaffinity columns (AflaTest, Vicam, Waters, MA, USA) connected to the glass syringes and vacuum system (2-3 mL/min flow rate). The columns were washed with 20 mL ultra-pure water, and targeted AFM 1 in the samples was eluted with 1 mL of methanol. Then, the samples were subjected to dryness on evaporation under the nitrogen flux (MultiVap-54), and the dried extracts were one-by-one reconstituted in a 1 mL solvent mixture of methanol/water (50:50, v/v).

Chromatographic Analysis
The determinations of AFM 1 were carried out on a high-performance liquid chromatography (HPLC) system (Shimadzu 10 VP, Kyoto, Japan) equipped with 10 AXL fluorescence detectors (Excitation at 360 nm and emission above 440 nm). A Kinetex C 18 column (Phenomenex, Torrance, CA, USA) 4.6 × 150 mm, 2.6 µm particle size, and an in-line filter of 0.5 µm were used. The isocratic mobile phase consisted of methanol/water/acetonitrile (6.4:28.1:10.5, v/v/v) with a flow rate of 0.50 mL/min.
Calibration curves with five points were prepared by diluting AFM 1 standard (Sigma ® , St Louis, MO, USA) in acetonitrile (CH 3 CN) at the concentration ranges of 2.5, 5, 10, 20, 40 ng/mL. The limits of detection (LOD) and quantification (LOQ) were calculated based on the signal-to-noise ratio (S/N) of 3:1 and 10:1. The LOD and LOQ values for AFM 1 in cheese samples under study were 0.017 and 0.055 µg/kg, respectively. All HPLC runs were carried out in triplicate and the data average values were expressed in the form of mean ± standard deviation.

Data Analysis
The results obtained from the trial were subjected to one-way analysis of variance (ANOVA) to determine differences among the cheese samples and the Tukey 5% test was applied. The significance level was accepted at the probability p < 0.05. All analyses were carried out using XLSTAT 2022 software (v.24.2.1300), with descriptive statistics and Microsoft Excel (v.14.0.4760.1000) also being used to summarize the data. Additionally, R version 4.0.5 software was used.