“Brazil nut,” “Pará nut,” or “Amazon nut” is the name popularly given to Bertholletia excelsa; these are fruits from nut trees sourced sustainably through extraction located in the Amazon region (1). Due to its nutritional content and the characteristics of the tropical forest in which it is found, with a hot and humid climate, this fruit becomes a suitable material for the growth of microorganisms, mainly fungi of the genus Aspergillus flavus (2).

Among the mycotoxin-producing fungi in BN, aflatoxins (AF) are observed with greater concern, as they cause disease and can lead to death (3). AF can cause harmful effects such as immunosuppressive, carcinogenic, mutagenic, and teratogenic effects, even in small amounts in food (4). Because they are microscopic substances that do not deteriorate the appearance of the food, it is difficult to detect them just by looking at the fruit. Studies point to the existence of four different types of aflatoxins common in Brazil nuts, which are AFB1, AFB2, AFG1, and AFG2 (1, 5).

Most of the places where these fruits are extracted are precarious and have poor hygiene conditions. For this reason, a sanitary orientation program and constant training in good production and manufacturing practices are necessary to guarantee the quality of the food (6). Health education, together with good production and manufacturing practices, is essential for the efficiency and safety of food products, being an important part of the process and a mandatory item within food safety legislation (5, 7, 8).

Ensuring a safe product with AF aflatoxin contamination within the limits of legislation is essential and a significant challenge for the productive and industrial chain, especially for BNs (7). In this sense, the relevance of this study is based on the use of advanced biomolecular techniques to evaluate the microbial profile through DNA sequencing and amplification of 16S and internal transcribed spacer (ITS) genes for bacterial and fungal analysis, respectively, and the presence of mycotoxins using HPLC-FD from different fractions of the nuts processing. Since the literature is often restricted to the genus Aspergillus, little attention is given to the microbial profile and the variation in its population over the production seasons.

In the HA, the bacteria identified in Shell_A are found in different environments, mainly in soil and decomposing material, as they have saprophytic characteristics. This result was expected since this fraction is in direct contact with the external environment and consequently more contaminated (1, 6, 16). On the other hand, Kernel_A and Cake_A showed a significant reduction in these bacteria, with only plastid DNA being detected in the majority, indicating low contamination of these fractions and adequacy in the good practices of processing these fractions.

When observing the Oil_A fraction, in addition to the expected plastid DNA, the presence of a greater proportion of different bacteria was observed, such as the genus Rothia, which are bacteria found in human skin and saliva but which could be emerging as opportunistic pathogens associated with various infections in immunocompromised individuals, drawing attention to the study of their diagnosis and symptoms presented by patients (17). In addition, the presence of the genus Staphylococcus in the oil, which is usually present in the respiratory cavity in humans and can act as pathogenic bacteria (18), may indicate contamination due to improper handling of these fractions during processing (19). Sphingomonas are bacteria found in different environments; however, their presence in Oil_A (HA) can be problematic since these bacteria are used precisely in bioremediation because of their ability to degrade oils. Associated with this, the presence of this bacteria, as well as the presence of fungi (Aspergillus, Fusarium, and Penicillium) in the samples, can greatly contribute to the degradation of this oily matrix, generating a product with unpleasant sensory and physical-chemical qualities (20–22). In addition, some species of this genus, such as Sphingomonas paucimobilis, an emerging opportunistic bacterium with a particular tropism for bone and soft tissue (23); nosocomial diseases (24, 25); meningitis (26); endocarditis (27); and some cases of pneumonitis caused by Sphingomonas ginsenosidimutans (28). On the other hand, in harvest B, in addition to bacteria, naturally occurring in different environments, such as soil and decomposing material, others from the genus Cronobacter were also identified. These bacteria are Gram-negative, facultatively anaerobic, opportunistic pathogens. Several species are resistant to desiccation and persistent in dry food products. They may even contribute to the deterioration of nuts, depending on the storage conditions. This is particularly relevant since nuts are predominantly consumed in this manner (29–31). Several bacteria identified in HB also have species described as human opportunistic pathogens which, as in the case of Pseudomonas, cause recurrent nosocomial infections and are also an important indicator of food deterioration, which is a danger because under conditions of low immunity or favorable concentration, they can trigger serious diseases, with many species described as highly resistant to antimicrobials in traditional use in medical protocols (32–34).

This information on bacteria is relevant, as most of the literature on nuts focuses only on bacteria from the microbiological standards of food in current Brazilian legislation (35) or on the classic fungal microbiota (5, 36–39). Regarding these, pathogenic microorganisms, such as Salmonella spp., were not detected in both harvests. Some samples presented Escherichia sp. in a very low proportion, except for the Oil_B sample, which indicated an abundance of 2.29% of this genus. One hypothesis raised is that this presence may indicate contamination of this fraction due to inadequate hygienic conditions at the site, corroborating the study by Arrus et al. (36). This genus of bacteria has different strains with different pathogens, for example E. coli, which can cause a variety of diseases (diarrhea, diseases associated with the gastrointestinal tract, and also co-infections such as meningitis and respiratory tract), and most of them are difficult to treat (due to observations of multiresistant to antibiotics), representing a greater health risk, and are often associated with diseases transmitted by food or water (40, 41).

Although the initial expectation regarding the oil fraction would be sterility or low contamination due to the low water activity of this food matrix, the exact opposite was observed. This is due to the richness and diversity of nutrients that this fraction has, thus creating a very favorable environment for microbial proliferation (42). de Oliveira et al. (43) found a similar situation and even proposed sterilization of nut oil through ozonation without impairing its physicochemical characteristics. The oil fraction was considered critical for both HA and HB, requiring greater intervention in good practices by handlers and the equipment used for extraction, transport, and storage.

The fungi observed in the samples are common in soils, with several considered phytopathogens, such as Alternaria and Cladosporium, while the genera Aspergillus and Penicillium are considered food degraders (44). These last two are the main fungal microflora and major producers of toxins that are harmful to plants, animals, and humans, with A. nomiae of great interest in Brazil nuts (16, 45). Fungi of the genus Alternaria, despite receiving little attention in the literature on Brazil nuts, are capable of producing toxins such as alternariol (AOH), alternariol methyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN), and altenuene (ALT), leading to food spoilage and posing a health risk to humans. This is a major concern for several countries in Asia and Europe (46–48). Freitas-Silva et al. (49) identified AME for the first time in Brazil nuts samples using liquid chromatography with tandem mass spectrometry (LC-MS/MS) without identifying Alternaria spp. in the analyzed samples. Our study showed that the DNA of Alternaria spp. in the two Brazil nut seasons showed that, depending on the climatic conditions, this phytopathogen/mycotoxin can constitute a potential danger for the Brazil nut.

The cake and oil fractions showed the highest amounts of aflatoxins. In the case of the cake fraction, this may be related to the higher prevalence of Aspergillus in this fraction; however, the same cannot be said for the oil fraction, as this had the lowest prevalence of Aspergillus among the fractions. This same inversion of prevalence in the proportions “fungi vs. toxins” was found and discussed by Álvares et al. (50) in nut samples from the region of the state of Acre (BR), where even without the significant presence of fungi of the Aspergillus type, the samples contained aflatoxins produced by them. A hypothesis that could be considered is the possible cross-contamination between the fractions at the time of extraction or preparation of the product, especially in the case of oil (Figure 2 and Table 1). Another possibility is contamination from the soil, as the presence of the fungus in the fruit is not a requirement for the production of mycotoxins (51, 52).

The amounts of aflatoxins found in this study's samples followed Brazilian legislation (35) and corroborated the results found by Álvares et al. (50). However, it is important to point out that even low, small amounts of aflatoxins can, in the long term, determine the triggering of serious diseases, especially those of the AFB₁ type, which is considered one of the most dangerous and may result in significant alterations in DNA methylation and hormones that can increase the susceptibility to various diseases, such as cancer, in addition to its hepatotoxicity (53–55). A study in mice showed that AFB₁ doses of 6 μg/g body weight could develop hepatocellular cancer in 90% of guinea pigs after 52 weeks (56). In this sense, it is important to point out that even with a small amount of toxin, an orientation for the nut portion that should be consumed safely is also necessary (3).

Although the Brazilian legislation (35) has exclusive indications of the content of aflatoxins for Brazil nuts and also for how this nut is presented (“with shell for direct consumption,” “shell for direct consumption,” and “without shell for further processing”), these fruits are usually sold in bulk, without industrialized packaging, and without refrigeration, which means they are outside the sanitary rigor. An alternative to this would be the need to place sanitary quality inspection and certification seals, as well as the dissemination of guidance material for cleaning these foods before consumption, since they are products that, despite being dry or toasted, can also be consumed in natura (6, 13, 57).

For many years, it was thought that the appearance of mycotoxins was a peculiarity of the storage and storage stages of fruits; however, studies show that their production can also occur during planting and plant growth (8, 16); therefore, hygienic-sanitary attention and care both in the stages that precede and those that proceed with extractivism must be reinforced and guaranteed (58). Another strategy that can be suggested for the control of aflatoxins, due to their low price and viability, is the early harvest of these fruits, as carried out by the study by Martins et al. (52) with specimens of peanuts where they were successful in this type of intervention or even the application of temperature-controlled packaging technologies for storage and distribution (59). The early detection of gene expression in fractions of Aspergillus species can be a valuable tool to predict the production of aflatoxin before its detection in Brazil nuts; however, these tools are not very accessible from a financial point of view (45).

Brazil nuts have a diverse microbiota, which is not restricted to fungi of the Aspergillus genus, as is most often the focus of most literature on nuts. Saprophytic, pathogenic, and enteric bacteria were also found in the samples in different proportions, showing a non-uniformity of the samples. Fungi of the genus Alternaria were identified in the analyzed samples, different from previous studies. The oil fraction presented microorganisms contrary to expectations regarding water activity and microbial proliferation. The microbiological and toxicological profile can be variable according to seasonality, extraction period, collection site, fruit handling, and processing. In addition, we must consider that health education is a valuable and necessary tool for mitigating microbial contamination. This needs to be implemented efficiently and supervised within the nut production chain. Guidance on consumption, previous care, the dangers of non-hygiene, and the correct preparation of these nuts are also necessary. Therefore, there is a need for the development of more laws, educational actions, and inspection programs, as well as the monitoring of continued studies on the fluctuation of microorganisms over the years and their correlation with climate studies. This is necessary to understand whether global climate changes are affecting this valuable nut of the Amazon rainforest.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by the Universidade Federal do Estado do Rio de Janeiro. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

VE: Writing – original draft, Writing – review & editing, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization. PM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. DC: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. OF-S: Writing – original draft, Writing – review & editing.