Keywords

15.1 Introduction

Mycotoxins are fungal secondary metabolites which may exhibit their effect as teratogens, carcinogens, mutagens, and estrogens. The presence of these toxins in foods may pose a serious health hazard to consumers and may lead to economic loss in food and feed industries. These include aflatoxins, patulin, ochratoxins, fumonisin, citrinin, trichothecenes, and zearalenone. In all these mycotoxins, aflatoxins are the most toxic and highly carcinogenic and therefore have a serious impact on human health (Nfossi et al. 2008; Paniel et al. 2010). Aflatoxins are produced by fungal species of Aspergilli, especially Aspergillus flavus and Aspergillus parasiticus (Nfossi et al. 2008; Paniel et al. 2010; Leong et al. 2010). Their contamination in food and feed has received public attention since few decades ago. Their presence in agricultural produce has great consequences on the economy of many affected areas mainly in the developing countries where there are poor pre- and post-harvest techniques (van Egmond 1983; Applebaum et al. 1982; Kumar et al. 2017). Up to now, 18 aflatoxins (AFs) have been identified, but only AFB1, AFB2, AFG1, and AFG2 are the most common of which among them, AFB1 is the most toxic (Hansmann et al. 2009). When a cow ingested aflatoxin B1 (AB1) from contaminated feed, enzymatic hydroxylation will transform it to aflatoxin M1 (AFM1), now classified as group 1 carcinogenic agent by the International Agency for Research on Cancer (IARC 2002; Krishnamachari et al. 1975).

Aflatoxins were first discovered in the 1960s in England when an outbreak of Turkey “X” disease killed around 100,000 turkeys and other farm animals. Heavy ingested peanut containing Aspergillus flavus was found to be the feed component that caused the disease (van Egmond 1983; Hansmann et al. 2009). In India, Rajasthan and Gujarat states also recorded a case of hepatitis that resulted in the death of about 106 people due to the intake of food containing aflatoxin (Bhat and Vasanthi 2003). Preliminary analysis confirmed the presence of Aspergillus flavus in maize which is the major food staple of these states (Kumar et al. 2017; Krishnamachari et al. 1975). Fungal growth and production of aflatoxins are generally found in tropical regions where there are high environmental conditions temperature, moisture, relative humidity, unseasonal rain during harvest as well as flood. Fungal proliferation in food is mainly due to bad harvesting practices, lack of good storage facilities, and poor conditions in transportation and marketing (Mohamadi and Alizadeh 2010; Matabaro et al. 2017).

15.2 Global Aflatoxin Occurrence in Food and Feed

As defined by CODEX Alimentarius (2011), any substances that are accidentally found in human food or feed of food-producing animals due to production, manufacturing, processing, preparation, treatment, packaging, packing, transport, or holding of such food or feed or as a result of environmental conditions are called contaminants. Contaminations in food and feeds with aflatoxins have a great negative impact economically and have received a lot of attentions since the previous decades. Global detection of these toxins in food commodities mainly in developing countries where pre- and post-harvest equipment are not enough to curtail the growth of these fungi is of great economic concern. Aflatoxins B1, B2, G1, and G2 occur naturally in foods and contaminate a large number of foods such as rice, wheat, corn, and peanuts (Schatzmayr and Streit 2013; Han et al. 2013) (Fig. 15.1). Table 15.1 shows recent investigations with different types of method performed globally for the detection of aflatoxins in food and feeds. The most toxic among them is AFB1 which is classified as group 1 liver carcinogen by the International Agency for Research on Cancer (IARC). Direct contact and indirect contact of human to AFs occur by consuming AF-contaminated foods and products from animals initially exposed to AF-contaminated feeds, respectively. Most developed countries set up a permissible level of AFs as low as possible because of their carcinogenic nature. A maximum permissible level of 2 μg/kg for AFB1 as well as 4 μg/kg for total AFB1, AFB2, AFG1, and AFG2 was approved by the European Union in a variety of products.

Fig. 15.1
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Different types of aflatoxins (source: Zhang et al. 2014)

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Table 15.1 Global occurrences of aflatoxins in food and feed in most countries
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The urgent need for control measures against toxicogenic fungi especially aflatoxins was suggested in a research conducted in Eastern Ethiopia when a high concentration of total aflatoxin level was detected in 93 out of 120 samples of groundnut analyzed using ELISA test. As per variation of total aflatoxin level, between 15 mg/kg and 11,900 mg/kg is an indication of its high occurrence in Ethiopian groundnuts which is by far beyond the limits set by the European Union (EU) (4–15 mg/kg), Food and Agriculture Organization of the United Nations (FAO) (15 μg/kg), and World Health Organization (FAO/WHO) standard (15 μg/kg) (Chala et al. 2013). From a survey of 200 feeds and 200 milk samples in China, 40% of the feed samples have AFB1 in the range of 0.05–3.53 mg/kg, while 36% were positive for AFB2 in the range of 0.03–0.84 mg/kg. Although the amount of aflatoxin B1 was slightly higher than aflatoxin B2 in the feeds, it was still below the acceptable limits of set aside by European Union 5 μg/kg as well as 10 μg/kg for China, respectively. The total amount of aflatoxins was also below the US acceptable limit of 20 μg/kg (Han et al. 2013). In Malawi, Matumba et al. (2014) collected samples of locally processed and imported maize as well as groundnut-based food products. The extent of aflatoxin contamination was analyzed with the help of immunoaffinity-reversed-phase liquid chromatography. All imported baby cereal foods and locally processed de-hulled maize have low contents of AFs below acceptable limit, while that of locally processed maize-based foods was above the EU maximum acceptable limit of 0.1 mg/kg; monitoring of AFs in locally processed foods will likely reduce AF amount and also reduce the risk of eating AF-contaminated food and feeds.

In Zimbabwe, fungal contaminations and aflatoxin were detected using high-performance liquid chromatography-fluorescence and standard mycology culture methods, respectively. Four out of six peanuts examined for fungal contamination were infected with Aspergillus flavus or Aspergillus parasiticus ranging from 3 to 20% of the seeds studied, while 27% of the peanut butter samples were also infected with either Aspergillus flavus or Aspergillus parasiticus. The result also indicated that, 91% of peanut butter and 17% of peanut samples are contaminated with aflatoxins with mean values of 75.66 ng/kg, respectively. It was found to exceed the EU acceptable level and hence advice sensitization for manufacturers so as to reduce contamination level (Mupunga et al. 2014). A study of 45 samples of ultra-high treatment (UHT) milk from the Indian states of Karnataka and Tamil Nadu were analyzed for the detection of aflatoxin M1 by reversed-phase HPLC using fluorescent detector. All the UHT milk samples tested were positive for AFM1, and 38% of these contained AF levels higher than the acceptable limit of 0.5 μg/kg prescribed by the Codex and Indian regulatory commission (FSSAI 2011; Siddappa et al. 2016).

In Lebanon, Elkak et al. (2012) conducted a research to detect AFM1 in 111 randomly selected cheese samples from local small dairy farms and dairy industries as well as imported cheese. From the cheese samples analyzed, AFM1 was detected in 67.56% of which AFM1 levels in 17.33% of the samples exceeded the European Commission (EC) acceptable limit of 250 ng/kg. Frequent supervision of locally processed cheese in Lebanon may drastically reduce the health risk associated with AFM1. Vagef and Mahmoudi (2013) provide an update on the level of AFM1 in 144 fresh and pasteurized milk samples from western region of Iran using ELISA technique. They concluded that the amount of AFM1 in both fresh and pasteurized milk was higher than the tolerable level of 0.5 ppb where the contamination level was significantly higher in winter than in summer. In a similar research, cow milk samples were found to contain AFM1 in an amount ranging from 0.01 to 1.2 mg/kg, out of which, 86.0% of the milk samples contained high quantity of AFM1 higher than the tolerable limit of 0.05 mg/kg set by European Union (EU) regulations. Other types of milk samples indicated a percentage of AFM1 as 80.0%, 60.0%, and 60.0% in goat, donkey, and breast milk, respectively (Kos et al. 2014).

In Brazil, a research was conducted to check the incidence and occurrence of aflatoxin M1 in cheese, yoghurt, and dairy drinks. A total of 123 samples were collected and analyzed using different methods in which all the samples tested have AFM1 higher than the acceptable level. Although there is lack of regulatory limit of aflatoxins in Brazil, this survey offered some useful information on the occurrence of AFM1 in Brazilian dairy products with potential risk to consumers as well as an insight into the need for establishment of Brazilian Maximum Residue Level (MRL) of AFs in food and feeds (Iha et al. 2011). Also in Croatia, Bilandzir et al. (2014) found a high concentration of AFM1 above tolerable limit when different animals were studied for the presence of AFM1 in their milk from July to September 2013. The result indicated that high level of this toxin in cow milk shows the use of contaminated feedstuff in some farms within the studied period.

15.3 Regulations

International and national regulatory bodies set maximum tolerable limits of aflatoxins as their carcinogenic and hazardous nature was detected in food and feed of humans and other animals, respectively. The joint FAO/WHO expert committee stated that the presence of aflatoxins in food should be limited to tolerable limit defined as the amount of a substance that cannot be removed from food or feed without discarding that particular food or compromising the exact availability of main food supplies. The first legal act on aflatoxin was established by United States Food and Drug Administration (USFDA) in 1965 when a maximum residual level (MRL) of 30 pg/kg for total aflatoxin (B1 + B2 + G1 + G2) was proposed. Since then, the maximum residual level has been regularly revised. The current regulations for aflatoxin approved by the Joint FAO/WHO Committee (CODEX), FDA, and some other countries are given in Table 15.2.

Table 15.2 Standard ranges of aflatoxins set by international organizations
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Many countries have set their maximum acceptable limit of aflatoxins in food and feeds. Industrialized nations set lower limit and regularly monitor and update their acceptable limit for import and export of food commodities likely to contain aflatoxin than developing and underdeveloped countries. In Table 15.3, for example, countries like China and the Philippines as reported by Anukul et al. (2013) set a tolerance level of 20 ppb for total aflatoxins in all human foods, while 30 and 35 ppb are the tolerance levels of aflatoxin B for all human foods in India and Malaysia, respectively. However, this difference of acceptable limit among countries brings about difficulties in the trades of commodities from one country to another. Even though in 2011, the Serbian Government has changed and harmonized their tolerable limit for AFM1 in milk with the European Union (EU) regulation, the 2013 occurrence of AFM1 in Serbian milk led to change in regulation where MRL was changed from 0.05 to 0.5 mg/kg. Kos et al. (2014) suggest permanent and harmonious regulations of AFM1 in milk and that of other aflatoxins in animal feeds with that of the EU taking into account that milk is one of the major food staples for Serbians.

Table 15.3 Maximum regulation limits for aflatoxins in food and feed in some countries
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15.4 Aflatoxin Detection in Food and Feed

15.4.1 Sampling and Sample Preparation

The main problem with aflatoxin analysis is obtaining a representative sample from the said commodity. A large amount of commodity may be required to increase the chances of toxin detection since a very small quantity may differ widely within any batch of food and feed. Detailed procedures for sampling and preparation of aflatoxin analysis can be found in FAO/WHO standard regulation 20/2015. Based on USFDA (2011) criterion, three steps which include sample size reduction, sample particle reduction, and sample homogenization for uniformity are vital in preparing any sample for aflatoxin detection.

15.4.2 Extraction of Aflatoxin from Food and Feed

An efficient extraction step is required in order to detect and quantify aflatoxins in any food samples. In polar protic solvents like methanol, acetone, chloroform, and acetonitrile, aflatoxins are normally and easily soluble. Aflatoxin extraction involves the use of organic solvents like methanol, acetonitrile, or acetone mixed in different proportion with a small quantity of water (Bertuzzi et al. 2011).

Determination of aflatoxins based on immunoassay technique requires extraction using mixture of ethanol and water. This is due to the fact that methanol has lesser negative effect on antibodies compared to acetone (Stroka et al. 1999; Lee et al. 2004). The clean-up technique which uses immune-affinity column (IAC) chromatography is usually followed after extraction (Ma et al. 2013). This immune-affinity column chromatography can bind with antigen and antibody with high reversibility and specificity which can separate as well as purify target analytes from matrices (Shelver et al. 1998). The crude sample is mainly applied to the column having a specific antibody to aflatoxins immobilized on a solid support like silica during clean-up. Aflatoxin binds onto the column and is retained with the sample moving beneath the column. The other washing step is usually required to remove impurities and unbound proteins when conducted with appropriate buffers and ionic strengths.

15.4.3 Trends in the Methods Used for Aflatoxin Detection

Over some decades, a lot of analytical methods and devices have been developed for the detection and separation of aflatoxins in food and feeds. Monitoring its presence in various commodities is important for the protection of consumer as well as producing raw materials prior to cost intensive processing or transport. Most of the devices or methods described below have advantages and disadvantages over one another in terms of their mode of operation, utilization, purchase, reliability, duration, and acceptability explained in many literatures.

15.4.3.1 Chromatographic Methods

A separation technique that involves the physical interaction between a mobile phase and a stationary phase is known as chromatographic technique. The separated components are to be distributed between mobile phase which is usually fluid passing along stationary phase (Braithwaite and Smith 2000). In practical point of view, the analyzed sample usually dissolved in mobile phase and applied as a spot on the stationary phase. Sorbents are the partitions between solid and liquid stationary phase when samples are analyzed in mobile phase. The most common chromatographic methods for evaluations of aflatoxins are described below.

15.4.3.1.1 Thin-Layer Chromatography (TLC)

De Iongh first used thin-layer chromatography (TLC) in 1964, and TLC was regarded as the best method for aflatoxin detection in 1990 by the Association of Official Analytical Chemists (AOAC). This separation technique usually depends on silica, aluminum, or cellulose as stationary matrix, while the mobile phase consists of a mixture of methanol, acetonitrile, and water immobilized on plastic or glass. Aflatoxin movement within these phases is based mainly on changes of aflatoxin solubility in the two phases. This technique has an application for the measurement of aflatoxins in agricultural produce and can also detect as small as 1–20 ppb of aflatoxin. It can however need a well-trained personnel and tedious sample pretreatment, and sometimes, it is not accurate since there is probability that errors may occur at multiple points along the process. This technique has application in measurement of already known aflatoxins at high concentrations, especially if new equipment is not readily available (Wacoo et al. 2014).

15.4.3.1.2 High-Performance Thin-Layer Chromatography (HPTLC)

This technique is like thin-layer chromatography, but unlike TLC, all the separation processes such as plate development, application of sample, as well as interpretation of data were carried out automatically in a precise and efficient way. This method is time-consuming and laborious and requires well-trained personnel and expensive instrumentations (Badea et al. 2004). Other limitations of this method are requirement of complex gradient mobile phase, large amount of organic solvent, and regular maintenance of equipment (Wacoo et al. 2014). However, some of these limitations were overcome by the use of gas chromatography.

15.4.3.1.3 Gas Chromatography (GC)

Gas chromatography mainly separates aflatoxins by the movement of carrier gas acting as mobile phase through the column acting as stationary phase that has a liquid coated onto inert solid particles (Cunha and Fernandes 2010). Electron capture detector (ECD) or flame ionization detectors (FID) are usually used to detect aflatoxins while gases are separated from other samples as they move along the column. Separation of aflatoxins in gas chromatography requires molecule derivatization to a detectable volatile form since most of the toxins are not volatile.

15.4.3.1.4 High-Performance Liquid Chromatography (HPLC)

Just like similar chromatographic methods, this method also separates aflatoxins as mobile phase moves along stationary one. This movement mainly consists of adsorbents depending on the chemical and physical structure of aflatoxins (Gamliel et al. 2017). The sample normally in liquid form moves along the column by carrier solvents where the aflatoxins separate from the main components during extraction. The procedure applied in this separation technique is usually differentiated from other techniques based on their column types and carrier liquid since only few detectors like ultraviolet and fluorescent light are coupled to HPLC.

15.4.3.2 Spectroscopic Methods

15.4.3.2.1 Fluorescence Spectrophotometry

This method can quantitatively measure from 5 to 5000 ppb of aflatoxin in 5 minutes and remains to be the most pivotal technique in the analysis and characterization of molecules that emit energy at a certain wavelength in peanuts and grains for aflatoxin analysis. As per Gamliel et al. (2017), derivatization may be required for best analysis of aflatoxin using fluorometry for improved aflatoxin fluorescence. As per approval by European High commission, aflatoxin detection limit using this method is moderately high than the limit of 4 μg/kg.

15.4.3.2.2 Frontier Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is the preferred method of infrared spectroscopy. In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is absorbed by the sample, and some of it is passed through (transmitted). The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. Like a fingerprint, no two unique molecular structures produce the same infrared spectrum. This makes infrared spectroscopy useful for several types of analysis (Mirghani et al. 2001) reported the use of frontier infrared spectroscopy for the analysis of aflatoxins in peanuts and peanut cake which use total internal reflectance. Detection of aflatoxins was also reported by Pearson et al. (2001) using transmittance and reflectance in single corn kernels.

15.4.3.2.3 Infrared Spectroscopy

Infrared (IR)-based methods require little sample preparation with extensive calibration. Near-infrared (NIR) and mid-infrared (MIR) are rapid and nondestructive analytical techniques and, hence, usually used for food quality (McMullin et al. 2015). Due to their limited sensitivity, commonly used IR-based method cannot detect mycotoxin directly in a food and plant tissues. However, it can be used as a detection tool following appropriate separation procedures like HPLC. The major limitation of this method is the limited sensitivity of detection.

15.4.3.3 Immunochemical Methods

According to Wacoo et al. (2014), this method of detection depends mainly on specific binding between antigens and antibodies. Various immunochemical methods have been developed because of the high affinity and specificity of antibodies on antigens. However, this binding specificity is not limited to antibodies and antigens but also on ligands and receptors which also have such affinity and high specificity (Sargent and Sadik 1999).

15.4.3.3.1 Radioimmunoassay (RIA)

Rauch et al. (1987) invented RIA with its application in the qualitative determination of insulin in human blood with further extension for of aflatoxin in contaminated food items. Langone and Vunakis (1976) confirmed through their studies the use of solid phase radioimmunoassay technique in determining aflatoxin B1 in peanut, and a limit of detection of 1 μg/kg was achieved. In addition, Rauch et al. (1987) reported the use of radioimmunoassays for the qualitative and quantitative determination of aflatoxin B1 and aflatoxin M1 levels. This immunochemical method depends on competitive binding among radioactive-labelled and nonradioactive antigens. Berson and Yalow (1968) reported that for a set number of antibody or antigen binding sites on the same antibody, radioactive-labelled antigen takes part with unlabelled nonradioactive antigen. A measured amount of labelled antigen and an unknown quantity of unlabelled antigen with that of standards react competitively with a known and small amount of the antibody. These labelled amounts of antigen are in inverse proportion to the amount of unlabelled antigen in the test. The advantage of this method is its capacity to run many examinations simultaneously with high reactivity and precision. One negative aspect of RIA is it requires antigen in pure form and used label isotopes associated with possible endangerment as well as problem associated with storage and disposing radioactive waste (Wacoo et al. 2014).

15.4.3.3.2 Enzyme-Linked Immunosorbent Assay (ELISA)

Avrameas (1969) invented the ELISA using enzyme-antigen conjugates and enzyme-antibody conjugates. ELISA method relies on the preciseness of antibodies for antigens, and the reactiveness of the assay is enhanced by tagging either the antibodies or the antigens with an enzyme that can be simply evaluated by use of specific substrates. Hence, an antibody which is immobilized onto a stable support may take an unlabelled antigen in the analyte, which is subsequently distinguished by a labelled antibody. As per Devi et al. (1999), mean immobilized antibody onto a solid support may capture an unlabelled antigen in the analyte, which later identified by a labelled antibody. ELISA method is presently used in the identification of aflatoxin in agricultural products (Anjaiah et al. 1989; Thirumala-Devi et al. 2002; Ondieki et al. 2014). Some commercially available ELISA kits that use enzymes alkaline phosphatase and horseradish peroxidase as labels in analysis of aflatoxins based on a competitive immunoassay format are extensively used (Ostadrahimi et al. 2014) and (Huybrechts 2011). The ELISA technique has advantages; that is, it is feasible to achieve the test on a 96-well assay platform resulting in the analysis of a large number of concurrent samples; the ELISA kits are cheap and easy to use, and most importantly, there is no possibility of health hazards associated with enzyme label.

15.4.3.4 Immunosensors

An international scientific endeavor, i.e., International Union of Pure and Applied Chemistry (IUPAC), defined a biosensor as any device that can provide précised quantitative and semi-quantitative interpretive data using biological understanding element in explicit connection with transducers (Shruthi et al. 2014). This device is based on interaction between biological components and transducers. Biological components such as enzymes, antibodies, and tissue slices are used to recognize and interact with a specific analyte, while transducers convert this interaction into a signal that can be amplified with respect to the concentration of the analyte (Shruthi et al. 2014). Amperometric, optical, potentiometric, magnetic, and colorimetric devices are normally used as transducers. Magliulo et al. (2005) reported a rapid and highly sensitive chemiluminescent enzyme immunoassay for the determination of AFM1 in a milk sample. Similar work using these transducers was described by Parker et al. (2009), Zangheri et al. (2014), Vdovenko et al. (2014), Mavrikou et al. (2017), and Stepurska et al. (2015). It is found to be highly sensitive, accurate, cost-effective, sensitive, and throughput in the screening of AFM1 as compared with other immunoassay. Cuccioloni et al. (2008) designed an assay for analytical test of aflatoxins B1 and G1 which is an alternate screening technique for mycotoxins. This determination approach to monitor toxins was based on surface plasmon resonance using neutrophil porcine elastase as bait. Its applications include moderate speed, recycling of the capturing surface and cost effective. Stepurska et al. (2015) has designed a potentiometric biosensor based on a pH-sensitive field-effect transistor and an enzyme acetylcholinesterase for the detection of aflatoxin B1 in real samples. It was proved to be very stable and highly sensitive when tested in the determination of AFB1 in walnut, sesame, and peanut. The application of protein for creating a highly sensitive site against AFB1 produced through bioimprinting as a means of detecting AFB1 by capacitive biosensors was reported by Gutierrez et al. (2016). This biosensor has the ability to generate specific interactions with aflatoxin B1 demonstrated in a linear relation between log concentration and signal registered of the target aflatoxin in a concentration ranges between 3.2 × 106 to 3.2 × 109 M when using ovalbumin as framework for bioimprinting. Other biosensors developed for aflatoxin detections include an aptamer for detection of AFB1 (Castillo et al. 2015) and an electrical immunosensor for detection of ultra-trace quantity of AFM1 in food products (Paniel et al. 2010). This immunosensor has a low detection limit of 0.01 ppb which is under the recommended level of 0.05 ppb and has good reproducibility.

15.5 Conclusion and Future Challenges

The occurrence of aflatoxins in food and feeds is of global concern both in terms of health implication and economic consequences especially in developing countries where pre- and post-harvesting practices are poor. These lead to the establishment of various regulatory bodies in different countries for the sole purpose of regulating and controlling risk associated with consumption of aflatoxins in food or feeds. Different analytical methods that are usually applied for identification of toxins in food and feeds include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC), which are largely time-consuming and expensive and require trained personnel and also a series of sample preparation. Based on these mentioned problems, it is important to develop better methods based on sensitivity and cost. This leads to the development of more improved analytical techniques that are rapid, more sensitive, specific, and on-site immunoassay. RIA and ELISA suffer a setback as they require pure state of antigen, used label isotopes associated with possible health hazard, and multiple washing steps, which sometimes prove laborious and time-consuming. The development of immunosensors like biosensors has brought about an opportunity for more rapid, highly sensitive, inexpensive and rapid on-site technique for easy detection of aflatoxins in food and animal feeds. This technique is based on interaction between biological components such as enzymes, antibodies, microorganisms, and tissue slices which recognize and react with a transducer like amperometric, optical, potentiometric, and magnetic devices. The biological component reacts and recognizes a specific analyte, while transducers convert this interaction into signal. However, these biosensors have some setbacks as some transducers are expensive and lose activity after some time except when stored under a better condition, require purification and isolation cost, and have slow response to time and longer recovery time.

Future research on recent recognition elements such as bacteriophages and aptamers should be focused where more robust, rapid, highly sensitive, cost-effective, and miniaturized biosensors for on-site detection of aflatoxins can be develop. The development of biosensors based on interactions between nanomaterials and biomolecules on the surface of nanofilms may also attract attention in future researches for aflatoxin detection in food and animal feeds.