Fungal Contamination of Nuts

#### **Chapter 4**

## Fungal Contaminants and Mycotoxins in Nuts

*Giulia Mirabile, Patrizia Bella, Antonio Vella, Vincenzo Ferrantelli and Livio Torta*

#### **Abstract**

Contamination by fungi and mycotoxins in nuts has achieved much attention in recent years. In fact, the fungal metabolites produced by the species of *Aspergillus*, *Penicillium* (aflatoxins and ochratoxins), *Fusarium* (trichothecenes, zearalenones and fumonisins) and *Alternaria* (alternariotoxins) with toxic and/or carcinogenic effects are considered a threat to human and animal health. In this chapter we will discuss the main fungal *taxa* and related mycotoxins most frequently associated with these materials. In this regard, the first results on the level of contamination by fungi and mycotoxins in samples of almonds and pistachios of different origins will be reported. The main strategies to reduce the risk of contamination will also be recommended.

**Keywords:** nuts, contaminating fungi, mycotoxins, *Alternaria*, *Aspergillus*, *Fusarium*, *Penicillium*

#### **1. Introduction**

Due to high nutritional value, pleasant taste and importance also in the pharmaceutical and cosmetic fields, nuts have been consumed by humans for thousands of years all over the world [1, 2]. Furthermore, some nuts are included in feed for several animal species. However, both the allergic forms to these products [3] and the health risks for prolonged and massive ingestion of mycotoxins present in these foods [4–6] are widely documented. Nut allergy is a hypersensitivity to some organic substances of this food caused by the consumer's immune system, whereas the risk of mycotoxicosis is related to the health of the nut. In particular, contaminating mycotoxins are produced by various saprophytic or phytopathogenic fungal microorganisms that can infect nuts and other foodstuffs throughout the whole production chain, from the farm to the plate [7].

Mycotoxins are a heterogeneous group of organic substances, generally characterized by low molecular weight, active at extremely low concentration (parts per billion, ppb, or parts per trillion, ppt; μg/L and ng/L, respectively), very resistant to degradation, produced by the secondary metabolism of numerous species of filamentous fungi (molds). When these microorganisms infect and develop on food and feed, they release the toxic molecules which, once ingested by the consumer (man or animal), can cause various acute (rash, vomiting, diarrhea, headache, etc.) or chronic (nephropathies, immunosuppression, carcinogenic effects, etc.) damages [8].

The health risks may arise from their carcinogenicity, so much so that the International Agency for Research on Cancer (IARC), based on epidemiological data, studies on cancer in experimental animals and mechanistic studies, has evaluated the carcinogenic risk of some mycotoxins in humans [9]. In order to protect the consumer from excessive exposure to the most dangerous mycotoxins, several Countries and federations of States have issued regulations that limit the maximum permissible content in a wide variety of foods for human and zootechnical use (for the EU, Commission Regulation (EC) No 1881/2006, Official Journal of the European Union, L. 364/5, 20.12.2006; Commission Regulation (EC) No 401/2006, Official Journal of the European Union, L70/12, 9.3.2006 and subsequent updates).

From the first report of a turkey disease ("turkey X disease") [10] associated with feeding these poultry with mycotoxin-contaminated peanuts (groundnuts), a large number of publications concerning the presence of fungi and their mycotoxins contaminating nuts have been spread all over the world. It is now known that among the mycotoxins most harmful to the consumer, listed by the IARC, are indicated aflatoxins, produced by fungi of the genus *Aspergillus*, ochratoxins produced by fungi of the genus *Aspergillus* and *Penicillium* and numerous others produced by different species of *Fusarium*, all capable of infecting nuts [7, 11]. Furthermore, several other mycotoxins, the "emerging mycotoxins" (alternariotoxins, enniatins, sterigmatocystein, etc.), produced by fungal species belonging to the aforementioned or to others genera (*Alternaria*), found in different nut samples, are currently being studied for their presumed dangerousness [12, 13].

#### **2. Mycotoxins and associated risk to human health**

The acute and chronic damages from massive and prolonged ingestion, inhalation or cutaneous absorption of mycotoxins are well documented and reported from all over the world.

From a historical point of view, the best known and described mycotoxicosis is ergotism (ergot of rye), widespread in some European regions since the Middle Ages. The disease is due to the ingestion of *Claviceps purpurea* sclerotia (rich in clavine alkaloids, lysergic acids, simple lysergic acid amides and peptide alkaloids), ground up with grains of various infected plants of cultivated and spontaneous species of the *Pooideae* subfamily (rye, millet, barley, oats, triticale, wheat, etc.). Two types of ergotism have been documented: convulsive and gangrenous. The symptoms included convulsions, hallucinations, skin burning and gangrene due to the constriction of the blood vessels, loss of hands and feets [14].

Several clinical studies have highlighted the correlation of mycotoxin effects with various pathological states in consumers, even if in most cases a "cause–effect" relationship is not clearly demonstrated [15]. Only for aflatoxins (in particular aflatoxin B1, AFB1) produced by some *Aspergillus* and *Penicillium* species, have been ascertained carcinogenic, hepatotoxic, teratogenic, and mutagenic effects on human health [16]. For this reason, IARC included the aflatoxins in the Group 1B: "Carcinogenic for humans" [17]. The risk of liver cirrhosis and immune effects, acute diseases such as hemorrhagic liver necrosis, edema and lethargy, up to death, have also been reported in frail individuals, children and in high-risk areas in developing countries [18–20]. Moreover, AFB1, once ingested by animals, is metabolized in aflatoxin M1 (AFM1) and excreted in the milk. Hence AFM1, included by IARC in the Group 2B, "Possibly carcinogenic to humans" [17], can be found in milk or milk products.

As regards ochratoxins, and in particular, ocrhatoxin A (OTA), carcinogenicity on laboratory animals is known, as well as damage to the kidneys, heart and liver injuries in humans. To date, there is no evidence of direct effects on the

#### *Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

appearance of carcinogenic phenomena on humans [21]. According to the IARC, OTA is included in the Group 2B [17]. OTA has been also associated with the Balkan endemic nephrophaty (BEN), disease affecting the rural populations of the Balkans and resulting in a high incidence of chronic kidney problems and cancers of the organs of the excretory system [22].

With reference to mycotoxins produced by fungi belonging to the genus *Fusarium*, the toxicological data indicate that fumonisins, trichothecenes (deoxynivalenol, T2 toxin, HT2 toxin) and zearalenone caused evident and known effects on humans, while others are considered a potential risk for consumer health. In particular, the fumonisins produced mainly by *F. verticillioides* and related species are suspected to cause esophageal cancer [23–26]. Fumonisin B1 is included in the IARC Group 2B [17].

Among the trichothecenes, deoxynivalenol (DON, known also as vomitoxin) produced by *F. graminearum* and related species, shows acute toxicity in animals (gastroenteritis, immunotoxicity, cardiotoxicity, refusal of feed, etc.) and poisoning in humans, with various symptoms such as nausea, vomiting, diarrhea and fever, as reported in China in the second half of the last century [27]. Because of the lack of data on its carcinogenicity in humans and only limited evidence on its carcinogenicity in experimental animals, DON is not classified by IARC [17]. T2 and HT2 (metabolized from the T-2 toxin after ingestion) toxins are considered agents of cytotoxic and immunosuppressive effects, which can cause acute intoxication and chronic diseases in both humans and animals. The fungal species most involved in the production of these secondary metabolites are *F. langsethiae*, *F. poae* and *F. sporotrichioides* [28]. The toxins derived from *F. sporotrichioides* are classified by IARC into Group 3 of carcinogenic substances [17]. Trichothecenes have been identified as the toxic agent in cases of Alimentary Toxic Aleukia (ATA, septic angina) associated with the consumption of moldy grain by both animals and humans in the USA, Japan, the former Soviet Union and elsewhere. The symptoms of the disease are characterized initially by general toxic stage (headache, weakness, fever, nausea, vomiting, gastroenteritis, etc.), followed by leukopenic stage manifested by changes in blood and, finally the angina-hemorrhagic stage [14].

Zearalenone (ZEA), mainly produced by *F. graminearum* and related species, does not cause acute poisoning in humans, but no studies on carcinogenic effects have been reported. ZEA caused an increased incidence of tumors in liver and pituitary cells in mice, but no carcinogenic effect was seen in rats, therefore considering limited carcinogenic effects in animals [17].

Aflatoxin, trichothecenes, ochratoxin A, fumonisins, zearalenone, fusarochromanone, have been shown to cause also immunosuppression and increase the susceptibility of animals to infectious disease [29].

Mycotoxins can pose several risks to human and animal health. The risks associated with health have often been characterized, however the mechanisms by which these toxins cause such damages have not yet well defined. Anyhow, the quantity and duration of exposure to mycotoxins, the synergy between the various fungal metabolites, the genetic predisposition and physical conditions of the consumer and other factors, can ultimately play a fundamental role in the manifestation of their toxic activity.

#### **3. Main mycotoxigenic fungal genera in nuts**

#### **3.1 The genus Aspergillus**

*Aspergillus*, a widespread Ascomycota genus belonging to the *Aspergillaceae* family, is divided in 6 subgenera and 27 sections with more than 400 species [30]. It received its name from Michieli in 1729 that, viewing the microscopic structure of its conidiophore, was reminded of the device called in Latin "aspergillum", used to sprinkle holy water during the liturgy [31]. *Aspergillus* species grow most as saprophytes on decaying vegetable organic matter, but they can also colonize human tissues, home and hospital environments. Furthermore, *Aspergillus* is one of the most important genera with high economic and social impact due to its ability to produce mycotoxins, dangerous secondary metabolites with potential carcinogenic activity against humans and animals [32]. On the other hand, some species are employed in several industrial and food production processes (production of citric acid, gluconic acid, kojic acid, amylases, cellulases, hemicellulases, soy sauce and miso) [33].

In this fungal *Taxon*, conidiophore is the most important microscopic structure for its identification. During mycelial growth, hyphae called "foot cells" form a single conidiophore perpendicular to cell axis. The conidiophore presents a large apex that form a rounded or elliptical vesicle (columella). In uniseriate species (*A. fumigatus*), the fertile area of the vesicle is surrounded by a layer of cell called phialides that produce long chains of conidia or conidiospores (**Figure 1a**). In biseriate *Aspergillus* species (*A. niger*) another layer of hyphae called metulae exist between the vesicle and the phialides. In some *Aspergillus* species (*A. flavus*), conidiophores can be uni- or biseriate. The vesicle with phialides, metulae if are present, and conidiospores form the "conidial heads" [32]. The size of conidiophore and conidial head, the presence of metulae, the shape and the color of conidiospore are important identifying features. All these structures are typical of asexual reproduction, but in *Aspergillus* sexual reproduction also occurs and its microscopic features (cleistothecia, asci, ascospores, Hülle-cells) are also important for identification. Macroscopically, colonies are granular or with a suede-like surface consisting of dens felt of conidiophores. Growth is usually very fast and mature colonies are white, yellow, yellow-brown, dark brown-black, green, gray or light blue in color (**Figure 2a** and **b**) [34].

*Aspergillus* species are thermophilic, preferring hot humid climates but can grow in a wide range of temperature (7–42°C). Spores are usually present in aerosol and can be easily dispersed by air for long distances. When they find optimal conditions of temperature and humidity, they germinate starting the colonization of the substrate. Most of *Aspergillus* species are saprophytes and soil inhabitants. Human and animal foods are ideal organic substrates in which, thanks to their enzymatic activity, they can grow degrading chemical components like hemicellulose, celluloses, pectins, but also fats and oils. They can contaminate major agricultural commodities before or after harvest, causing decay in storage or disease in plants. Although acid pH and low amounts of water normally do not support fungal growth, most of *Aspergillus* species are able to grow at these conditions, colonizing foods and nuts [35].

During the last decades of '900 the discovery of toxins produced by *Aspergillus* associated with poultry and other domesticated animals' deaths all over the world, raised new awareness that *Aspergillus* were very dangerous for both human and animal health [36, 37].

There are 4 groups of aflatoxins (AFs): aflatoxins B1 and B2, aflatoxins G1 and G2. Other mycotoxins, as M1 and M2, originating from their metabolism in humans and animals, can be found in milk and dairy products [38]. Aflatoxins are the most important and dangeroustoxins produced by *Aspergillus* in food, feed and nuts, especially in peanuts. They are produced mostly by some strains of *A. flavus* and *A. parasiticus*. In recent years other species were classified as aflatoxigenic, like *A. bombycis*, *A. ochraceoroseus*, *A. nomius* and *A. pseudotamari*, but compared to the first two mentioned, they are found less frequently in foodstuffs [39].

*Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

#### **Figure 1.**

*Microscopic characteristics of* Aspergillus *sp. (A),* Penicillium *sp. (B),* Fusarium *sp. (C),* Alternaria *sp. (D). cn = conidia; Ph = phialides; v = vesicle; m = metulae, s = stipe; b = branch; mc = macroconidia; ps = pluriseptate conidium of* Alternaria *sp. Scale bar: A-C = 10 μm; D = 20 μm.*

Other secondary toxic metabolites produced by *Aspergillus* species and in particular by *A. niger* and *A. ochraceus*, are ochratoxins. This group includes ochratoxin A (OTA), ochratoxin B (dechlorinated OTA) and ochratoxin C (ethylated OTA). Among them the most studied for its high diffusion and its toxicological importance is OTA. This toxin, has nephrotoxic effects in humans and animals and has carcinogenic action [38, 40].

#### **3.2 The genus** *Penicillium*

*Penicillium* is one of the most common fungi occurring in a wide range of habitats with worldwide distribution and high impact on economy, human and

**Figure 2.** *Nuts contaminated with* Aspergillus flavus *(a),* A. niger *(b),* Penicillium *sp (c),* Fusarium *sp (d).*

animal life. Its name derives from its conidiophore shape, with a brush-like appearance (a penicillus). It belongs to *Aspergillaceae* family and it's divided in 32 sections with almost 500 species; the teleomophs are ascribed in ascomycetes genera, as *Eupenicillium* and *Talaromyces* [30]. In nature, Penicillium species live decomposing organic materials. On the contrary, other species of *Penicillium* are widely used in the food industry for the production of cheeses such as Roquefort and fermented sausages [41].

For *Penicillium* identification the conidiophores are of great taxonomic importance, resulting from the different branching of the stipe. Depending on the species, in fact, there may be only phialides at apex of stipe, producing conidia, metulae and phialidia, or different order of metulae and phialides. In particular, conidiophore shapes range from being simple to quarter-verticillate depending on the branching levels between the stipe and metulae and phialides. Conidia, typically globose, are produced in long chains by phialides and, when mature, show different colors (**Figure 1b**). Macroscopically, colonies appear of velvety consistency, they are fast in growth and with color varying from green to light blue (**Figure 2c**) [41, 42].

Ecologically, *Penicillium* species live as saprophytes showing optimal growth at low and moderate temperature ranging from 5 and 37°C. Some species colonize decaying vegetation, other are specialized in infecting food commodities. *Penicillium* spores are consistently spread by airborne dispersion and contaminate a large variety of organic substrates. Rapid fungal colonization and intense enzymatic activity cause the spoilage of infected materials. *Penicillium* can invade plants before harvest or during storage. They are also capable to grow during drying process, colonizing substrate poor in water, like nuts [43, 44].

*Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

In addition to damages caused in fruits and vegetables by *Penicillium* colonization, some species are able to produce a great variety of mycotoxins. These include ochratoxin A, patulin, citrinin, penicillic acid, cyclopiazonic acid, citreoviridin, roquefortine C and other secondary metabolites. Ochratoxin A and patulin are the most studied due to their worldwide diffusion and their implication in animal and human diseases [45]. In particular, exposure to these two toxins results in mutagenic, teratogenic, neurotoxic, genotoxic and nephrotoxic effects or acute effects like nausea and gastrointestinal damages. The major producers of ochratoxin A in nature are *P. verrucosum* and *P. nordicum*, while *P. expansum* is the major producer of patulin [46]. While patulin is commonly detected in fresh fruits like apple and its derivates, ochratoxin A can be found in a large number of food products including nuts [47].

#### **3.3 The genus** *Fusarium*

The anamorphic fungi belonging to genus *Fusarium* are ubiquitous, growing in soils but also in living or dead plants and in several commodities. These ascomycetes, ascribed to *Hypocreaceae* family and mainly included in the genus *Giberella*, currently contains several complexes species. Some of them, in particular those contaminating commodities, produce several mycotoxins with acute and chronic effects on human and animal health [48, 49].

Microscopic characteristics such as macro-, microconidia and chlamydospores of *Fusarium* are distinctive for genus identification. Macroconidia, produced from phialides sometimes gathered in sporodochia, are long, slender, dorso-ventrally curved (spindle-shaped), pluricellular and with a basal foot cell near the attachment point to the phialide (**Figure 1c**). Is this particular shape of macroconidia that defines the genus. Microconidia, formed from aerial mycelium are little propagule, mono or bicellular, smooth, hyaline and ovoid or cylindrical in shapes. Chlamydospores are overwintering structures, rounded, produced vegetatively from mycelium, able to resist to unfavorable conditions and germinate under the onset of suitable conditions. Macroscopically, colonies are wooly or cottony, flat and presents several colors such as white, cream, tan, salmon, cinnamon, violet, purple (**Figure 2d**) [50, 51].

Fungi of the genus *Fusarium* generally prefer cool climates and have a complex ecology. As saprotrophs, they live in the soil, on crops or on decaying organic material. Many species are primary pathogens in field, capable of causing vascular diseases. The most affected crops in the field are wheat and corn, but infection by these fungi can also occur in vegetables, nuts, ornamental plants, trees and foodstuffs during post-harvest storage [7, 52, 53]. Some *Fusarium* species produce dangerous mycotoxins that cause cellular toxicity, effects on animal growth and development and human cancer [54]. The main toxic classes produced by *Fusarium* include trichothecenes such as T-2 toxin, deoxynivalenol (DON) and nivalenol (NIV) produced mainly from *F. sporotrichoioides*, *F. graminearum* and *F. culmorum*; fumonisins B and B2 produced mainly from *F. fujikuroi* and *F. proliferatum*; zearalenone produced from *F. culmorum*, *F. graminearum* and *F. crookwellense* [55].

#### **3.4 The genus** *Alternaria*

The genus *Alternaria* belongs to the Ascomycota family of *Pleosporaceae* and it is divided into 26 section and approximately 300 species (*Lewia* is the most known sexual stage), characterized by saprophytic and pathogenic species causing plants diseases, postharvest damages and humans' allergies [56]. Some species of *Alternaria* also produce several mycotoxins in infected plants and foods [57].

Macroscopically, the colonies are flat, downy to wooly and the surface is grayish-with at the beginning and greenish-black or olive-brown at maturity [58]. Characteristics of *Alternaria* is the production in chains of dark-colored and multicelled conidia ovoid to oblcavate, with longitudinal and transverse septa and a beak tapering apical cells (**Figure 1d**) [59, 60].


#### **Table 1.**

*Most recurrent toxigenic fungi isolated from some nuts reported in recent studies.*

*Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

*Alternaria* spp. colonize a wide range of plants and growth as saprophytes in plant residues, in soil or as fungal pathogens, colonizing mostly fruits and herbaceous plants and it is diffused in humid environment characterized by temperature ranging from 18 and 32°C [61]. *Alternaria* spp. can produce toxic metabolites that play a fundamental role in fungal pathogenicity and food safety. Today are known about 70 alternaria-toxins, some of them very dangerous for humans and animals [62], including alternaiol, alternariol monomethyl ether and tentoxin commonly founded in substrates like tomato, oil seeds, wheat, blueberries and walnuts. Toxicological data about mycotoxins produced by *Alternaria* are very limited but they have been shown to have cytotoxic, fetotoxic and teratogenic effect on animals [63]. Recent studies focused on emerging groups of mycotoxins produced by *Alternaria* species, described as potentially hazardous [64].

All the fungi mentioned above were frequently isolated from nuts all over the world (**Table 1**).

#### **4. Detection methods of contaminating fungi and mycotoxins in nuts**

In the last decades several laboratory methodologies were defined both to detect, enumerate and isolate fungi from nuts and to evaluate the presence of single specific mycotoxins and their respective concentrations. However, so far, only few methods can be considerate "official" and validate by Public Authority.

To apply isolation techniques, is necessary to take a representative sample to submit to the laboratory test and, also for this, the official regulations provide the correct methods of sampling, handling and pretreatment of the samples.

To detect, enumerate and isolate contaminating fungi from sampled nuts two cultural-based methods are most frequently applied: the dilution plating [85] and the direct plating [86]. In the first case, unshelled or shelled nuts are finely chopped or homogenized in a mixer and suspended in water or water containing 0.1% peptone. Serial decimal dilutions are spread on agarized nutrient medium in Petri dishes (plates). In the second case, nuts particles (or whole seeds) are placed directly on media, eventually after surface sterilization in sodium hypochlorite.

The agarized artificial nutrient media most employed are:

PDA (Potato Dextrose Agar), also known as the "universal substrate", it is mainly used to stimulate the growth and sporulation of filamentous fungi;

SDA (Sabouraud Dextrose Agar) is the medium normally used for the primary isolation of fungi, often with the addition of antibiotics. This medium allows the detection of the "standard" morphology, limiting the development and sporulation of fungal colonies;

DG18 (dichloran-glycerol agar), recommended for the evaluation of CFU and the isolation of yeasts and molds from dry and semi-dried foods, including fruits, spices, grains, nuts, meat and fish products [85];

DRBC (Agar Dichloran Rosa Bengal Chloramphenicol), promotes the selective growth of molds and yeasts present in food [87];

PCA (Agar Plate Count) is an agar medium that allows the non-selective growth of molds, yeasts and bacteria;

OGYE AGAR BASE, contains yeast extract, useful for the growth and UFC count of molds in clinical, food and dairy product samples.

Media recommended for *Aspergillus* and *Penicillium* identification include Czapek Yeast Autolysate agar (CYA) and Malt Extract agar [34, 41]. More characters useful for other taxonomic characters can be obtained by using other media such as

Czapek's agar (CZ), Yeast Extract Sucrose agar (YES), Oatmeal agar (OA), Creatine Sucrose agar (CREA), Dichloran 18% Glycerol agar (DG18), Blakeslee's MEA and CYA with 5% NaCl [34, 41].

Specifikke nutrient-arme agar (SNA), potato dextrose agar (PDA) and YES agar can be used for *Fusarium* identification while dichloran rose Bengal yeast extract sucrose (DRYES) agar and potato carrot agar (PCA) are suggested for *Alternaria* species [88, 89].

All plates are incubated at temperatures ranging from 25 and 30°C for up to 7–9 days and, every 3 days, observations are made on the number and type of fungal colonies grown. Higher incubation temperatures are useful to distinguish between species [34, 41].

In the methodology of the dilution plating the total number of fungal colonies (colony-forming units, CFU), referred to the relative decimal dilution, is used to calculate the level of fungal contamination of the analyzed sample. Moreover, observations under the stereoscopic microscope and the optical microscope allow to identify the genus of belonging of the colonies. It is therefore possible, on the total of the CFUs detected, to determine the percentage of the different mycotoxinogenic genera.

In direct plating analyses, results are usually expressed as percentage of infected particles/nuts.

Among the grown fungal colonies, some of the most representative, because probably belonging to mycotoxigenic species (yellow and black aspergillia, green and blue penicilli, *Fusarium* spp., etc.) are first transferred into plate containing agarized medium and purified to be submitted to identification tests and other analysis.

Although culture-based methods for detection and identification of fungal toxin producing fungi are widely used, they showed some limitation due to timeconsuming and labour-intensive aspects. It also requires facilities and mycological expertise.

For this reason polyphasic approach based on morphological, physiological and molecular methodologies is suggested for a more accurate identification of mycotoxin fungi producer [89]. Morphological analysis, macro and microscopic observations are made, also growing the isolate on suitable media and at different temperatures. Macroscopic features such as shape, color, texture and speedy growth of the colonies, among others, and microscopic characteristics as mycelial structures, spores, conidia are often fundamental taxonomic characters to identify the species [31, 34, 41, 90–92].

It is not uncommon, however, that these characters are not sufficient for exact identification. In order to avoid mistakes it is possible to consider some physiological parameters (enzymatic activities, secondary metabolites production, etc.) that can provide further data for better identification [93]. It is always good practice, in any case, to support and confirm the identification of the fungal isolate with appropriate DNA based molecular analyzes. A DNA marker for reliable species identification is the internal transcribed spacer rDNA area (ITS) now accepted as the official barcode for fungi [94]. However, this locus is insufficient for correctly identifying some species and other possible secondary markers include 'nuclear large ribosomal subunit' (LSU rDNA) [95], the 'nuclear small ribosomal subunit' (SSU rDNA) [96], 'β-tubulin (BenA)' [97], 'elongation factor 1-α (EF-1-α)' [98] and the 'second largest subunit of RNA polymerase II (RPB2)' [99].

The identification of fungi using molecular markers is improved after DNA isolation from mycelium of pure and axenic cultures [100]. A great variety of DNA extraction methods are available [101]. Commercial kits and customized methods are typically employed. Critical to the successful isolation of nucleic

#### *Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

acids is the cell disruption step with an appropriate buffer (typically CTAB) and techniques that assure high quantity and quality of nucleic acids and no release of potential PCR inhibitors. After this step, nucleic acids can be purified to eliminate impurities [102]. Species-specific PCR methodologies targeting conserved genes or regions of taxonomical interest or by focusing on the mycotoxigenic genes have been extensively applied for identification of mycotoxigenic fungal contaminants [103–106].

In order to detect and quantify mycotoxins in nuts there are several analytical techniques, but official controls are carried out with screening methods, using immunoenzymatic techniques (ELISA) and with confirmatory methods as highperformance liquid chromatography with fluorescence detection (HPLC-FLD) coupled to tandem mass spectrometry (LC–MS/MS). In most cases, methods are validated on a single matrix and do not meet the supervisory bodies' needs that require different mycotoxin limits for different matrices. Regarding extraction method, solid-phase extraction (SPE), solid–liquid extraction (SLE), and liquid–liquid extraction (LLE) are common techniques used for LC–MS/MS analysis for mycotoxin. The challenge of mycotoxin analyses is that they have different proprieties and polarity. Therefore, the right choice of the extraction method can be difficult.

More recently new techniques were developed to detect low-level mycotoxin contamination, to reveal the presence of "masked mycotoxins", complex food matrices in which the mycotoxin contamination occurs or to evaluate the co-occurrence of more mycotoxins in the same sample [6].

#### **5. Strategies to limit contaminating fungi and mycotoxins in nuts industrial chain**

Fungal and mycotoxins contamination occurs both in field (pre-harvest), during harvesting and post-harvest management [107]. In particular, aflatoxigenic aspergilli are of significant concern in terms of consumer health.

Generally, in field, several factors not easily controllable, can have a significant impact on fungal infection, including: crops damages due to drought or to insect, delay in harvesting, extreme weather events such as heavy rains, and or sudden frosts. Moreover, it is well known that any management practice to maximize plant performance and decrease plant stresses will decrease fungal and AFs contamination. Proper agronomic practices (tillage, fertilization regimes, right plant density and irrigation), guaranteeing the best vegetative development of dried fruit plants, can represent a valid means of prevention. Different cultivars of nuts show different susceptibility to *A. flavus* and AF accumulation. The conversion of orchards into more resistant cultivars is one possible measure of control [108–111].

Moreover, control of parasitic insects or other biotic adversities can limit the development of contaminating fungi, able to settle in the fruits through the lesions they cause [112].

During the harvesting process, great care must be taken to maintain the integrity of the product, avoiding contact with the ground or with other materials with a high risk of contamination. The damaged products are certainly those most susceptible to contamination during the subsequent phases.

Transport and storage are two important stages to be monitored [68]. Drying should take place soon after harvest and as rapidly as possible, because the prolongation of temperature, humidity and ventilation conditions favorable to the development of microorganisms, can lead to irreversible contamination of the product by both fungi and mycotoxins.

Nuts differ in their storage requirements in function of oil and fatty acid compositions. Temperatures ranging from 4 to 15°C, kernel moisture content around 2.5%, aw of about 0.7, or relative humidity below 80%, oxygen concentration below 2.5%, and dark conditions are ideal storage conditions for most tree nuts [107, 113].

In post-harvest some physical and chemical methods aimed at decontamination from aflatoxins can be effective for their control. Removal of visibly damaged nuts by manual or mechanical sorting prior to processing significantly reduces AF contamination, limiting the number of potentially contaminated nuts in subsequent processing steps [114]. Other processes, although not very convenient from a practical point of view, can allow to reduce the concentration of AF, such as chemical (ammonia) or thermal (peanut roasting) treatments. Some other technologies, such as irradiation and improved packaging materials, can also minimize post-harvest aflatoxin contamination [115].

However, the best solutions to reduce aflatoxin contamination and improve both economic sustainability and food safety, are the integration of pre- and post-harvest technologies.

In order to protect the health of consumers and ensure the fairness of international trade, FAO and WHO have developed a code of good practices to help contain the phenomenon (www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1& url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStan dards%252FCXC%2B59-2005%252FCXP\_059e.pdf).

These practices are also applied to all nuts and are summarized in the following points:


In addition to these practices, some studies have highlighted the possibility of using biological control strategies for the control of mycotoxinogenic fungi [112].

The definition of programs to monitor fungal and mycotoxin contamination in processed nuts are essential to ensure the food safety for the consumers and an effective scientific system for the control of mycotoxin levels is represented by the HACCP method (Hazard Analysis and Critical Control Point).

This method is based on the preventive analysis of hazards and on the control of socalled critical points detected during the production and manufacturing process [116].

The HACCP strategy is quite simple, at least in theory, as it considers all the steps of the supply chain, from the choice of the cultivars to the finished product ready for marketing and defines the control protocols on each phase of the process.

#### **6. A study case: fungal contaminants and mycotoxins in almonds and pistachios in Sicily (Italy)**

In last years (2016–2021) at the Experimental Zooprophylactic Institute of Sicily (Palermo, Italy), analyzes on contamination by total aflatoxins or AF B1 have been

#### *Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

carried out on 618 samples of pistachios, from both Sicilian and foreign origin, using the validate HPLC method coupled with fluorescence detection (FLD). Out of this large numbers of controls, only 7 samples were positive for total aflatoxins, whose contamination ranges were included between the values 0.2 and 1.7 micrograms per kg, lower than those required by European regulations.

In order to acquire information also on the state of contamination by mycotoxinogenic fungi, an investigation aimed at the isolation and identification of any fungal microorganisms present in both pistachio and almond samples was recently carried out. The samples were analyzed at the laboratories of the Department of Agricultural, Food and Forestry Sciences of the University of Palermo, for mycological tests and at the Experimental Zooprophylactic Institute of Sicily, for toxicological tests.

Shelled almond and pistachio samples were collected from different warehouses located in Southern Italy and analyzed in order to determinate total fungal contamination and the frequencies of toxigenic genera (*Aspergillus*, *Fusarium*, *Penicillium* and *Alternaria*). Isolation and enumeration of fungal colonies were carried out employing the dilution plating methodology [85] on PDA. All plates were incubated at 25±1°C for 9 days and the total fungal contamination was evaluated every 3 days. *Aspergillus*-like colonies found growing in the isolation plates were subcultured into PDA and monoconidial pure cultures were assigned to their corresponding section or species based on macroscopic and microscopic characteristics.

The macro-morphological features recorded were colonies diameter, color, shape and texture. Microscopic observations were performed using an Axioskop (Zeiss, Oberkochen, Germany) microscope coupled to an AxioCam MRc5 (Zeiss, Oberkochen, Germany) digital camera. The microscopic characteristics recorded were dimension of conidial heads, vesicle shape and diameter, presence of metulae, size and shape of phialides and conidia. Finally, morphological identification was carried out using taxonomic keys.

Although the toxicological tests on the samples of nine walnuts excluded the presence of aflatoxins, first results showed that fungal contamination in almond and pistachio samples ranged from 2.5x10 to 2.6x104 and 2.5x10 to 1.5x103 CFU/g respectively. The genera *Aspergillus* and *Penicillium* were recovered at high frequencies both in the pistachio and almond samples with significantly differences within the samples. Occasionally, other fungal genera were isolates (*Mucor*, *Rhizopus*, *Paecilomyces*, etc.), while no *Alternaria* or *Fusarium* colonies were observed. *Aspergillus* was the most predominant fungal genus both in almond (77%) and pistachio (89.8%) samples. *Penicillium* spp. frequencies ranged from <1 to 15.9%. These data are comparable to those reported in similar studies both in relation to the level of total fungal contamination an as regard the diffusion of the two considered genera [72, 74].

According to morphological characteristics, *A. flavus* and *A. niger* were identified in pistachio samples, whereas no *A. flavus* was isolated from almonds samples.

#### **7. Conclusion**

The problem of mycotoxins has become one of the aspects that most affect the nuts market, even if the real problem is represented by mycotoxinogenic fungi that can contaminate these commodities. Fungi infecting nuts in field can rapidly grow and produce mycotoxins during storage when conditions are suitable. Preventing AFs and other mycotoxins accumulation in the finished product can be achieved by either controlling the contaminating fungi or mycotoxins production in pre- or

post-harvest stages of production, by using any of several measures alone or in combination [108–111].

However, the more articulated and complex the production chain is, the greater is the risks and possibilities of contamination by fungi and their mycotoxins. Furthermore, although the concentration of the most dangerous aflatoxins may be very low or completely absent, some mycotoxigenic fungi may be present and in favorable condition can produce these secondary metabolites making the food no longer safe for the consumer.

In order to guarantee, products healthiness, mycotoxins detection should be supported by mycological analysis throughout the entire supply chain. Another appropriate practice should be to investigate the presence of a greater number of mycotoxins, given the great biodiversity of fungi potentially capable of contaminating nuts.

Several guidelines, therefore, have been developed to identify critical points in the production process and to define strategies aimed at directly and indirectly reducing the production and spread of mycotoxins.

Contamination prevention is necessary because most detoxification methods require testing and are not completely approved by the industry or follow all safety requirements. Quality assurance systems for tree nut industries, including supplier qualification, are essential to prevent the presence of toxigenic fungi and prevent the consequent production of toxins along the production chain.

Moreover, there is a significant interest in considering the impact of climate changes on mycotoxin-producing fungi during pre- and post-harvesting [117].

#### **Author details**

Giulia Mirabile1 , Patrizia Bella1 , Antonio Vella<sup>2</sup> , Vincenzo Ferrantelli<sup>2</sup> and Livio Torta1 \*

1 Department of Agricultural, Food and Forestry Sciences, University of Palermo, Palermo, Italy

2 Experimental Zooprophylactic Institute of Sicily, Palermo, Italy

\*Address all correspondence to: livio.torta@unipa.it

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Fungal Contaminants and Mycotoxins in Nuts DOI: http://dx.doi.org/10.5772/intechopen.100035*

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### **Chapter 5**

## Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew Nuts: Its Implications on Health

*Modupeade C. Adetunji, Stephen A. Akinola, Nancy Nleya and Mwanza Mulunda*

#### **Abstract**

Edible nuts are popular worldwide based on their varied attributes such as desirable taste, high nutritional value as well as some health benefits. Globally, the most popular edible nuts are groundnuts or peanuts, almond, cashew nut among others. Due to the rich nutritional composition of nuts, they tend to be prone to contamination by toxigenic fungi which could ultimately results in the release of fungal metabolites known as mycotoxins into nuts. In view of the nutritional composition of nut and its high susceptibility to fungal attack, this chapter looks at the nutritional profile, mycotoxigenic fungi and aflatoxins contamination of peanuts, cashew nuts and their products with a central focus on Africa where the effect of aflatoxin contaminations is more prominent.

**Keywords:** public health, peanut, cashew nut, aflatoxigenic fungi, aflatoxin, mycobiota diversity, food safety, Africa

#### **1. Introduction**

Edible nuts are popular worldwide based on their varied attributes such as desirable taste, high nutritional value as well as some health benefits. Moreover, production of edible nuts can be done under various growing conditions and climates [1], however edible nut production is mainly in the tropical and subtropical regions of the world.

Globally, the most popular edible nuts are groundnuts or peanuts (*Arachis hypogaea*) and are classified as legumes [2] with several other tree nuts such as almond (*Prunus dulcis*), cashew (*Anacardium occidentale*), Brazil nut (*Bertholetia excelssa*), hazelnut (*Corylus avellana*), macadamia (*Macadamia integrifolia*), pecan (*Carya illinoinensis*), pine nut (*Pinus pinea*), pistachio (*Pistachia* V*era*), and walnut (*Juglansregia*) [1, 3]. Although nuts are considered as food with numerous health benefits, they are prone to contamination by toxigenic fungi which could ultimately results in the release of fungal metabolites known as mycotoxins into nuts [4].

Contamination of food commodities by mycotoxins has become a global food safety concern [5, 6]. Aflatoxins are secondary metabolites produced by members of the genus *Aspergillus* mainly *A. flavus*, *A. parasiticus* and *A. nomius* [7]*.* This genus is very ubiquitous and is known as a primary inhabitant of soil that contaminate a variety of agricultural commodities especially cereal grains and oil seeds including nuts [8]. Consumption of aflatoxin contaminated food results in a condition known as aflatoxicosis. The severity of aflatoxicosis symptoms ranges from vomiting, abdominal pains and liver damage in acute aflatoxicosis as a result of ingesting large doses of the toxin. Whereas ingestion of smaller doses leads to chronic aflatoxicosis which is asymptomatic and may result in hepatocellular carcinoma [9]. There over 20 aflatoxins but only four of them occur naturally i.e. aflatoxins (B1, B2, G1, G2). These aflatoxins are identified based on their fluorescence (B-blue or G-green) under ultraviolet light [10].

Aflatoxin contamination has led to reduced international markets especially in the developed nations, thereby advocating for stringent measures requiring import products to have very low concentrations of aflatoxins. Since nuts are used as food as well as food ingredients, regulatory limits have been established and set at 4 μg/kg for total aflatoxin (B1 + B2 + G1 + G2) and ≤ 2 μg/kg for aflatoxin B1 by the European Commission [11, 12]. Implementation of good manufacturing practices in the nut production chain is very important so that the nuts comply with limits of the importers [13]. As a result, many countries have conducted research on the diversity of aflatoxigenic fungi as well as the extent of aflatoxin contamination in edible nuts as well as their products to ensure that their produce meets the required standards [14]. This chapter looks at the nutritional profile, mycotoxigenic fungi and aflatoxins contamination of peanuts, cashew nuts and their products with a central focus on Africa where the effect of aflatoxin contaminations is more prominent.

#### **2. Nutritional profile of peanut**

Peanut (*Arachis hypogaea* L.), groundnut or monkey nut (**Figure 1**) is the fourth oilseeds crop and 13th food crop grown worldwide because of its nutritional, medicinal and economic values [17, 18]. Several delicacies could be prepared from peanut ranging from products like roasted peanuts, peanut butter, peanut oil, peanut paste, peanut sauce, peanut flour, peanut milk, peanut beverage, peanut snacks (salted and sweet bars) and peanut cheese analog (**Figure 2**). Raw peanut is subjected to different processing which could alter or determine the nutritional composition of the end products. Processing such as roasting of peanut enhance its colour, flavour, taste, aroma and crunchy texture [20]. It also reduces the bacteria load and aflatoxin-producing fungi in raw peanut [21].

**Figure 1.**

*Showing (a) the groundnut plant in the field, (b) harvested plant and (c) unshelled and shelled groundnuts [15, 16].*

*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

#### **Figure 2.**

*Various forms in which ground nut can be consumed, adopted from market insider with modifications [19].*

#### **3. Proximate composition of peanut**

Peanut is an excellent source of nutrients similar to other nuts with a substantial amount of lipid, protein and fibre content along with some amount of carbohydrate, vitamins, and minerals. The nutrient constituents and content of peanut have been previously reported; protein 20.7%–25.3%, crude fat 31–46%, ash 1.2%–2.3%, crude fibre 1.4%–3.9%, carbohydrate 21–37%, and moisture 4.9%–6.8%) [22]. Nuts including peanuts provide over 10% of the recommended dietary allowance of nutrients (protein, iron, thiamine and vitamin E) for adult males [23]. The nutritional composition of peanut is shown in **Table 1**.



**Table 1.**

*Nutritional composition of groundnut.*

#### **4. Nutritional benefits of peanut on human health**

The major components of peanut are Protein, fats, and fibre (**Table 1**), and are present in their most beneficial forms. The protein is plant-based, while the fat is unsaturated, and the fibre is composed of a complex carbohydrate that are beneficial for human nutrition.

#### **4.1 Peanut protein**

The nutritional value of a food protein is determined by its essential amino acid contents and its digestibility. The protein content in the cake could reach 50% after the peanut oil has been extracted [24]. Peanuts contain all the 20 amino acids in variable proportions [25]. According to its Protein Digestibility Corrected Amino Acid Score (PDCAAS) peanut proteins and other legume proteins such as soy proteins are nutritionally equivalent to meat and eggs and ideal for human growth and health [26]. The true protein digestibility of peanuts is comparable with that of animal protein [27].

#### **4.2 Fatty acid composition of peanut oil**

Although peanuts and tree nuts have high lipid contents, peanut oil is rich in unsaturated fat, predominantly, monounsaturated fats (MUFA which have been associated with lower cardio-vascular risk) [28]. The MUFA of the regular US peanuts is 49–57% while a medium (66–69%) and high oleic (78–80%) rich peanuts have been reported [29]. The consumption of MUFA promotes arteryclearing which keeps the flow of blood and lowers the risk of atherosclerosis, heart attack or stroke [30]. Clinical studies demonstrated that intakes of MUFAs and PUFAs are associated with low risk of cardiovascular diseases (CVD) and death, whereas saturated fat and trans-fat intakes are associated with high risk of CVD [28].

#### **4.3 Dietary Fibre**

There are soluble and insoluble dietary fibres which have health benefits such as lowering the risk of heart diseases, diabetes and maintenance of a healthy weight [31]. Other health benefits includes, the lowering of blood cholesterol, improvement of bowel movement and reduced risk of metabolic syndrome [32]. The dietary fibre content of dry roasted peanut was reported as 8.4 g per 100 g of peanut similar to that in soybean (9.3 g per 100 g) while the total dietary fibre of defatted peanut flour (15.8%) was comparable to that of defatted soybean flour (17.5%) [33]. This substantial amount of dietary fibre could help individuals reach their recommended daily allowance of 38 g for men and 25 g for women.

*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

#### **4.4 Vitamins and minerals**

Peanut has been recognised as a great source of niacin, which is important for the proper functioning of the digestive systems, skin and the nerves. It helps in the conversion of food to energy and supposed to protect against Alzheimers disease and cognitive decline [34]. Peanut is an excellent source of vitamin E whose consumption in good quantities could lead to benefits against coronary heart disease [35]. Peanut also contains good amounts of folates which are important during infancy and pregnancy for the production and maintenance of cells. It was reported that a 100 g of peanut can provides the Recommended Dietary Allowance [36] levels of copper (127%), manganese (84%), iron (57%), phosphorus (54%) and magnesium (42%) intake which is associated with reduced inflammation [37, 38] decreased risk of metabolic syndrome [38] and type II diabetes [39]. They are also referred to as a nutrient dense food, rich in multiple natural micronutrients (**Table 1**) including bioactive compounds such as resveratrol, phytosterols, phenolic acids and flavonoids that are beneficial to health. This makes them a viable option for improving the nutrition status of the malnourished, neonates, growing, or those in need of critical nutrients [40].

#### **5. Health benefits of peanuts**

Peanuts lipid profile is high in unsaturated fatty acids than the saturated fatty acids, trans- fat-free, cholesterol-free. Its low saturated fats thus qualify it as safe and desirable. Apart from basic benefits of daily nutrition, peanut consumption leads to long term health benefits such as the management of cancer [41], effective weight management [42] and lower body Mass Index [43] and management of hunger [44].

#### **6. Cashew nut**

Cashew (*Anacardium occidentale* L.) is one of the most traded processed tree nuts on the global market [45] and has become a source of income to people in the producing countries through export markets [13]. Cashew nuts are among tree nuts which are known to possess many health benefits such as reduced chances of cardiovascular diseases, diabetes, metabolic syndrome, weight gain and obesity as well as mental instability [46, 47]. This is mainly because tree nuts contain several nutrients needed for the proper functioning of the human body such as unsaturated fatty acids, proteins, mineral, vitamins, phenolic compounds and fibre [47, 48].

Following an analysis of raw cashew nuts from Brazil, India, Vietnam, East and West Africa, Rico, Bulló [47] highlighted fat as the major constituents of cashew nuts with an average of 48.3% of its total weight. However, in another study, Abubakar, Abubakar [46] reported a much higher percentage of total fats (56.4%) in raw cashew nuts from Nigeria. Nevertheless their findings were within the range (40–57%) reported in literature [49]. Fats are known to play several roles in the diet such as provision of energy, essential fatty acids as well as fat soluble vitamins [50].

The cashew tree produces the nut (kidney shaped) as the main fruit and an accessory fruit known as the cashew apple (**Figure 3**) [52, 53]. The main traded cashew products are the raw nut, the seed and the cashew nut shell liquid (CNSL) whereas the apple is converted into various beverages [54].

**Figure 3.**

*Showing (a) cashew tree, (b) developing fruit, (c) cashew nut and apple, (d) roasting of shelled nuts, (e) raw shell nuts [51].*

#### **7. Contamination of peanuts and cashew nuts by toxigenic** *Aspergillus* **species**

Despite the fact that developing countries cover the world's greatest peanut producing area, yield is very low especially in Africa because of poor socioeconomic factors as well as the prevailing weather conditions [55]. The climatic conditions in the tropics and subtropics promote the proliferation of diverse pathogenic fungi capable of producing aflatoxins [56–58]. Extensive research on aflatoxins began in the 1960s with analysis on peanuts after poultry deaths in the United Kingdom, Kenya and India. This death was linked to the consumption of contaminated peanut meal [59]. The analysis of mould contaminants in peanut meals from various countries, implicated *Aspergillus flavus* [58] as the chief mould contaminant. *Aspergillus* species are ubiquitous group of soil borne fungi that can be found in agricultural soils, storage and food processing facilities as well as in the distribution systems [7, 60]. *Aspergillus parasiticus* is a soil inhabitant normally associated with pod and seed contamination whereas *A. flavus* thrives well in aerial environment and are therefore more likely to contaminate tree nuts [61]. Nuts can be colonised by fungi before harvest (**Figure 4**). Several authors have reported the presence of diverse *Aspergillus* species in the groundnuts and cashew nuts (**Table 2**).

Initially aflatoxin production was linked to *A. flavus* [85], therefore most researchers use *A. flavus* as an indicator of possible aflatoxin contamination in food commodities. For example, Sultan and Magan, [11] isolated several *Aspergillus* species from *Flavi, Circumdati* and *Nigri* sections, but performed aflatoxin producing potential tests only on the isolates from section *Flavi.* Similarly, Oyedele and co-workers [77] also isolated three *Aspergillus* species (*A. flavus, A. parasiticus* and *A. tamarii*) from peanut samples. However, determination of the toxigenicity of the isolates was carried out on those identified as *A. flavus* only. In another survey by Riba and colleagues [64] isolated species belonging to *Flavi, Circumdati, Terrei* and *Nigri* sections from shelled peanuts. Despite the fact that species from section *Nigri* had the highest incidence of occurrence, toxigenicity tests were done only for isolates from *Flavi* section. However some authors have reported aflatoxin production by species outside the section *Flavi* [86–89]. Therefore, it is important for researchers to determine the aflatoxigenicity of all isolates even if they do not belong to the *Flavi* section.

The high prevalence of the black *Aspergillus* i.e. section *Nigri* has been reported in many peanuts samples. Mohammed and Chali [55] reported the prevalence rates of *A. niger* ranging from 35 to 66% in peanut samples from the fields, storages and

*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

#### **Figure 4.**

*Colonisation of peanuts by Aspergillus at different production stages, (a) showing pods and kernels already contaminated before harvesting, (b) contaminated unshelled peanuts and (c) contaminated shelled peanuts in storage [36, 62, 63].*



#### **Table 2.**

*Incidence of Aflatoxigenic Aspergillus spp. in peanuts and cashew nuts from Africa.*

in market places. According to Riba and colleagues [64], *A. niger* is mainly used in fermentations. Riba and associates [64] also reported high incidence of *A. niger* in analysed peanut samples. Although there are no reports on aflatoxin production by *A. niger,* there have been reports on ochratoxin A production by some *A. niger*

*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

strains [90, 91]. On the other hand, Akinola and co-workers [92] highlighted the possibility of gene transfer between toxigenic and atoxigenic strains in feedlots resulting in strains previously known as non aflatoxigenic becoming toxigenic. Acquisition of aflatoxin producing genes by *A. niger* may become a threat to the fermentation industry where it is mainly used.

The quality of the edible part of cashew nut is of great importance as it is used as either as food or ingredients in processed products. However, like others cashew nuts are also prone to fungal contamination with consequent mycotoxin production. **Table 2** shows the presence of *Aspergillus* species especially the section *Nigri* being present in most of the samples. Lamboni and others [65] highlights the implication of this section in food deterioration. The same author reported production of mycotoxins by some isolates from cashew nuts in Benin belonging to the section *Nigri* such as *Aspergillus tubingensis, A. niger, A. brasiliensis, A. carbonarius, A. luchuensis, A. aculeatus, A. aculeatinus*, however none of the isolates were able to produce aflatoxins in culture. A similar observation was noted by El-Samawaty and colleagues [93] where the majority of the *Aspergillus* species in cashew nuts from Saudi Arabia belonged to section *Nigri.* Adeniyi and Adedeji [94], emphasised the importance of moisture in the kernels during storage as it may contribute to the colonisation of the nuts by mycotoxigenic fungi.

#### **8. Aflatoxin contamination of groundnut and cashew nut**

Aflatoxin B1 is the most potent and commonly produced naturally occurring aflatoxin [95], hence its presence in most of the samples and the most reported by researchers (**Table 3***).* African countries especially those in Southern Africa namely Botswana [98], Democratic Republic of Congo [100, 101], Malawi, Mozambique [56], South Africa [101], Zambia [123, 124] and Zimbabwe [125–128] had the highest incidence of aflatoxins in the samples analysed. Most batches had more than 50% of the samples testing positive to one or more of the naturally occurring aflatoxins (**Table 3**).

In samples from Northern Africa, aflatoxins were detected though incidences of contamination were less than those reported in Southern Africa. Most samples from the batches analysed had less than 50% aflatoxin contamination occurrence rate with a few exceptions like Sudan in peanut butter. Weather conditions in Northern African countries are characterised by high temperature and humidity [129], due to being surrounded by the Mediterranean Sea and the Atlantic Ocean which promotes growth, occurrence of toxigenic moulds and subsequent aflatoxin production [130].

From West African States, Nigeria and Benin had majority of their peanuts and cashew nuts contaminated with aflatoxins and with almost 100% incidence rates. Total aflatoxin contamination in peanut cake from Benin exceeded the stipulated EC regulatory limit (4 μg/kg) whereas in cashew nuts the concentration was below the limit of detection (LOD). Studies have shown that peanut production in Africa is faced with so many challenges such as poor resources and field management which resulted to the nut being neglected. Peanuts harvest and marketing are often delayed thereby exposing the peanuts to high levels of aflatoxin contamination [95]. Contamination of nuts by aflatoxin can either be before or after harvest and a gradual increase in aflatoxin content with prolonged storage [131] could be expected. In an analysis of aflatoxin levels in peanuts at farms and markets in Uganda, Kaya and co-workers [132] reported the presence of aflatoxins at farm level in ≥60% of the peanuts. Results from analysis of raw peanuts, peanut flour, roasted peanuts and peanut butter [73, 109] showed a



#### *Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*


*Nuts and Nut Products in Human Health and Nutrition*


*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

> **Table 3.**

*Aflatoxin contamination of groundnuts, cashew nut and their products in African countries.*

decrease in aflatoxin concentration in the order raw peanuts ˃ peanut flour ˃ roasted peanuts ˃ peanut butter. These results are in agreement with those of Siwela and colleagues [133] who reported a 51% reduction in aflatoxin after roasting of peanuts during large scale peanut production.

Peanut oil is the most utilised oil by people in the tropics due to its affordability [134, 135]. Analyses of peanut oil samples have shown the presence of aflatoxins especially in the unrefined oils. It has also been reported that oils extracted from peanuts often have higher aflatoxin contamination [136]. Abalaka [113], highlighted the use of crude oils by the majority of the Nigerian population hence their exposure to aflatoxins [113]. Most of the analyses in oils were from Nigeria and Sudan. Sudan is known as a major vegetable oil producer.

Cashew nut production in Africa is dominated by West African countries [137] hence the high number of reports from this quarters. Most of the studies in Africa were from Nigeria as it is one of the leading producers of cashew nuts worldwide. The result of analyses on aflatoxins in cashew nuts showed that they were within the EU and FDA regulatory limit of 15 μg/kg for total aflatoxin in nuts intended for further processing. However Milhome and associates [14] highlighted some samples from Brazil having total aflatoxins greater than this limit.

#### **9. Impact of Aflatoxin on health**

Mycotoxins finds their way into human and animal body through the consumption of mycotoxin contaminated foods [138]. Mycotoxins causes significant decline in animal productivity and general health performance. Out of the over 400 mycotoxins identified in food and animal feeds, those capable of causing significant health effect in humans and animals includes aflatoxin (AFs), fumonisins (FUM), zearalenone (ZEA), T-2/HT-2, deoxynivalenol [139] and ochratoxin A [15], they are of great concern for their effects on animal and human health [140]. Aflatoxins are naturally occurring chemical contaminants of foods such as cereals, legumes and nuts; groundnuts and cashew nuts [141], *Aspergillus parasiticus* and *Aspergillus flavus* are the primary producers of aflatoxin in crops [142]. The consumption of contaminated peanuts and cashew nuts which serves the function of food ingredients and as snacks could results in mycotoxicosis in humans and animals. Similarly, the detection of aflatoxin in animals carcases have been related to the ingestion of aflatoxin contaminated feed ingredients such as peanut [143]. Aside food substrates, mycotoxin occurrence have been reported in animal feed, animal feedlots and animal derived food products [92, 144, 145]. Aflatoxin is regarded as the chief of mycotoxins based on their degree of toxicities [146]. They have been classified as class 1 human carcinogens based on their deleterious effect on the health of both humans and animals [147, 148].

Human exposure to aflatoxin occurs due to the consumption of contaminated agricultural produce and animal derived food product [143]. Aflatoxin ingestion have been reported to cause teratogenic, mutagenic, carcinogenic immunosuppressive, hepatotoxic, nephrotoxic, and genotoxicity effect in humans and animals [144]. The degree of toxicity of mycotoxins on health of animals or humans is a function of the aflatoxin type, species and sex [149]. The liver is the major target organs for aflatoxin toxicity and could show symptoms such as liver lesions and tumour upon exposure to low and moderate doses of aflatoxin [146, 150]. The consumption of aflatoxin contaminated nuts could impair the immunity, feed efficiency and cause a teratogenic and mutagenic effect in animals [151–153]. The consumption of this nuts could pose a threat to consumer's health causing ill-health, *Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

immunosuppression, cancer and liver and kidney damage in humans and animals [150]. The consequences of aflatoxicosis accounts for more than 40% of the diseases in developing countries [9]. In tropical and subtropical countries with less or lack of regulatory activities governing the acceptable level of aflatoxin in food and feeds, the risk of human aflatoxicosis is huge. [154, 155]. The study of Ibeh *et al.* [156] reported the effect of aflatoxin on male fertility. In their study, males with high aflatoxin levels in their serum had abnormal sperm morphology, motility and sperm count. There are also evidence to the transfer of aflatoxin from mothers to babies. Authors [157–159] have reported the detection of aflatoxin M1 in breast milk of mothers exposed to aflatoxin contaminated foods in Gambian and United Arab Emirates respectively. Neonatal jaundice was reported in foetus exposed to aflatoxin in Nigeria and Iran [160, 161]. Studies have also shown the negative effect of aflatoxin on birth weight, gestational age, birth height, in blood samples obtained from mothers [158, 159].

Yousef and Lamplugh [162] have also reported mobility and mortality cases as a result of aflatoxin ingestion in humans. Chronic aflatoxicosis can results from the continuous exposure to aflatoxin contaminated foods and could cause reduction in life expectancy, cancer, immunosuppression and stunting in children [154].

#### **10. Aflatoxin regulation**

After the discovery of aflatoxins and their effects on health of both humans and livestock, regulatory limits were set in the late 1960s [163]. The United States of America was the first country to set the aflatoxin limit of 20 μg/kg [164] and the EU limit (4 μg/kg) for total aflatoxin and 2 μg/kg for aflatoxin B1. However there was harmonisation of aflatoxin standards for the EU countries which took place in 1997 and implemented in 1998. Total aflatoxin for peanuts needing further processing which was previously set at 10 μg/kg was changed to 15 μg/kg and 4 μg/kg for nuts intended for human consumption. Aflatoxin B1 was set at 8 μg/kg and 2 μg/kg for nuts that required further processing and direct human consumption respectively [165]. Not all countries adopted the harmonised standards, for examples in Asia, China and the Philippines limit of aflatoxins set by these countries were 20 μg/kg while Indonesia set 15 μg/kg for aflatoxin B1 [166].

#### **11. Conclusion**

Nuts and nut products and specially peanuts and cashew nuts have long been recognised for their nutritional content and contribution to good health. One of the limitations to the role of these nuts in human nutrition and health is their susceptibility to *Aspergillus* species and related aflatoxins. Aflatoxins have continued to be a problem from the time of discovery as their presence in nuts especially in African countries is still above the limits. Nuts are sources of livelihood to most people in developing countries as they can be used in nutrition as well as for income generation but its high susceptibility to aflatoxin contamination poses a huge threat to the consumers as well as reducing its value economically especially at the International market [139]. As aflatoxins are not really a threat to the developed countries, because of their stringent rules on acceptable limits in foods meant for human consumption.

This chapter revealed that a larger percentage of the nuts produced in Africa are contaminated with aflatoxin concentration above the regulated permissible level, hence consumers of nuts especially peanuts in Africa are at risk of aflatoxicosis

despite the nutritional importance of the nut. Hence, strategies to reduce the proliferation of aflatoxigenic fungi in nuts while on the field and at post-harvest level should be harnessed. Some suggested strategies to achieve this includes:


It is recommended that future research should focus on the nutritional advantages of peanuts and cashew nuts and their related health benefits beyond the ones that have been identified in this chapter.

*Nutrient Composition and Aflatoxin Contamination of African Sourced Peanuts and Cashew… DOI: http://dx.doi.org/10.5772/intechopen.95082*

#### **Author details**

Modupeade C. Adetunji1 \*, Stephen A. Akinola<sup>2</sup> , Nancy Nleya3,4 and Mwanza Mulunda<sup>3</sup>

1 Department of Biological Sciences, Trinity University Yaba, Lagos, Nigeria

2 Department of Microbiology, North West University, Mmabatho, South Africa

3 Department of Animal Health, Northwest University, Mmabatho, South Africa

4 Department of Applied Biology and Biochemistry, National University of Science and Technology, Bulawayo, Zimbabwe

\*Address all correspondence to: modupe.adetunji@trinityuniversity.edu.ng

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 4
