Aflatoxins and Other *Aspergillus*-Related Metabolites

#### **Chapter 8**

## Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care

*Nur Azura Mohd Said, Noor Sheryna Jusoh, Norhafniza Awaludin, Mohammad Rejab Ismail, Noor Fadilah Mohd Bakri, Lily Suhaida Mohd Sojak and Faridah Salam*

#### **Abstract**

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are potent mycotoxins produced by the fungal genus *Aspergillus*. Their occurrence in grain corn is alarming hence the need for rapid on-site detection. An immuno-based biosensor technique for detection of the aforementioned toxins is described here. Highly specific in-house polyclonal antibodies against AFB1 and OTA were employed as bioreceptors in a label-free electrochemical biosensor; immobilized on modified screen-printed carbon electrodes (SPCEs). The immuno-functionalized SPCEs were first characterized on a laboratory electrochemical workstation for proof-on-concept study using differential pulse voltammetry (DPV) electrochemical technique. An Android-based device is improvised as a portable electrochemical reader integrated with internet of thing (IoT) features which include cloud server and a dedicated website. Sensitivity achieved by the modified SPCEs on the portable device is superior compared to enzyme-linked immunosorbent assay (ELISA) method and lab-based electrochemical workstation. The miniaturized biosensor system has been successfully tested on cornfield for in-situ mycotoxins detection with simple sample extraction. Analysis performed on twenty samples were validated using chromatographic analysis. This biosensor-IoT system offers a potential application for real-time detection and the portable reader serves as an excellent tool for point-of-care in routine monitoring of harmful mycotoxins.

**Keywords:** aflatoxin B1, ochratoxin A, electrochemical biosensor, immunosensor, point-of-care, portable electrochemical device

#### **1. Introduction**

#### **1.1** *Aspergillus* **spp. and mycotoxins**

*Aspergillus* is an oligotrophic fungus that can grow in an aerobic environment and is capable of producing a harmful secondary metabolite known as mycotoxin. In agriculture, mycotoxins contamination is one of the perturbing global food safety issues that need to be addressed. Its occurrence is widely spread infecting various important crops and commodities, [1] both during pre and postharvest conditions, and forages [2]; besides contaminating food products and animal feed. Among major crops that are susceptible to mycotoxin contaminations are peanuts, corn, rice, sorghum, chili, millets, and legumes [1]. Of recent, mycotoxins incidence in aquaculture also have been extensively reported, including fish feed [3, 4]. In food chain, storage environments such as poor aeration and high humidity are among factors that trigger and accelerate the growth of fungi, which consequently leads to mycotoxins production. Therefore, mycotoxins can exist in the field before harvest, postharvest, or during processing, storage, and feeding.

Among the most commonly observed mycotoxins that posed concerns to human health and livestock are aflatoxins, ochratoxin A, fumonisins, zearalenone, and deoxynivalenol. The harmful impacts of the mycotoxins have long been observed in both humans and animals [5]. Exposure to even low concentrations of mycotoxins in long term has been associated with liver diseases such as cancer, hepatitis, and jaundice, and to the extent of being carcinogenic. Aflatoxins and ochratoxins are principal toxins produced by *Aspergillu*s spp. Human exposure to mycotoxins can happen either directly or indirectly. Directly, we are susceptible to mycotoxins when obliviously consuming grains, cereal-based products, or food contaminated with the toxigenic fungus. Indirectly, on the other hand, mycotoxins can enter human food chain *via* compromised animal feed. In terms of economic impact, farmers with contaminated crops or sick animals will lose their income and agribusiness will be greatly affected [6]. The prevalent issue of mycotoxins still remains the biggest challenge for animal feed producers and therefore, regular monitoring of mycotoxins is imperative.

#### *1.1.1 Aflatoxins*

The term aflatoxin is derived from the name of its main fungal producer that is *Aflavus*. Aflatoxins, also commonly known as a postharvest mold, are listed as potent mycotoxins. They are mainly produced by *Aspergillus flavus* and *A. parasiticus*. Other aflatoxin-producing species, albeit less frequently encountered, include *A. nomius*, *A. bombycis*, *A. pseudotamari,* and *A. ochraceoroseus*. Grains or food products contaminated with aflatoxins are not safe to be consumed. These toxins are resilient and stable against any thermal, physical, and chemical treatments along the feed chain production. In farm animals, aflatoxicosis can affect the liver and interrupt the digestive system. This in turn has a negative impact on livestock production with a reduction in body weight and feed conversion rate. Aflatoxins can contaminate a variety of livestock feeds and cause enormous economic losses, estimated at between US\$52.1 and US\$1.68 billion annually for the U.S. corn industry alone [7]. Even more unfortunate, aflatoxin in the animal's body system can be carried to eggs, meat, milk, and organ tissues. Indirect consumption of aflatoxins through food by humans can also cause toxin poisoning such as imperfect growth, weakened immune system, and liver damage, and have the potential to cause cancer and death.

#### *Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

There are more than 20 types of aflatoxins, but the four main types are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). The terms B1, B2, G1, and G2 are addressed based on their fluorescence under ultraviolet light where B denotes blue and G denotes green color. Both AFB1 and AFB2 are produced by *A. flavus* while AFB1, AFB2, AFG1, and AFG2 are produced by *A. parasiticus* isolates. Meanwhile, aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) are hydroxylated metabolites of AFB1 and AFB2, respectively (**Figure 1**). Among all these types of aflatoxin, AFB1 has been classified as a class 1 human carcinogen by the International Agency for Research on Cancer [8]. The order of toxicity of aflatoxin is as follows: AFB1 > AFG1 > AFB2 > AFG2 [9]. Aflatoxins have been reportedly found in peanuts, cattle feed, liquid milk, cashew nuts [5], and also grain corn [10].

#### *1.1.2 Ochratoxin*

There are three classes of ochratoxins, namely ochratoxin A (OTA), ochratoxin B (OTB), and ochratoxin C (OTC) (**Figure 2**). They are produced by *Aspergillus* and

**Figure 2.** *Chemical structures of the ochratoxins.*

*Penicillium* species, mainly *A. ochraceus*, *A. carbonarius*, *A. niger,* and *P. verrucosum*. Among these, OTA is considered the most abundant and harmful mycotoxin. OTA has been shown to be a potent nephrotoxic, hepatotoxic, and teratogenic compound. OTA has also been categorized as group 2B carcinogen for humans by the International Agency for Research on Cancer [8]. The intake of feed contaminated with OTA affects animal health and productivity and may result in the presence of OTA in the animal products. In acute cases, death may occur due to acute renal failure hence this toxin is regarded as nephrotoxic [11].

Ochratoxin commonly occurs in agricultural commodities, especially cereals this is corn and various foods such as cereal-based, wine, tea, coffee, cocoa, herbs, milk and milk products, poultry, fish, pork and eggs, fruits and vegetables, beans, dried products, infant foods as well as for poultry and other animal feeds. OTA is mostly found in the cereal grains such as maize, barley, oats, wheat, rye, etc. In a recent study, the abundance of ochratoxin A in grain corn is increasing in subregion of Asia in the second quarter of 2021 [10].

#### **2. Mycotoxins and their detections**

#### **2.1 Current state detection for mycotoxins**

Hitherto, instrumentation methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas spectrometry (GC) are regarded as gold standard for food contaminants analysis, including mycotoxins. The chromatography system is usually coupled with detectors such as fluorescence, ultraviolet–visible, or mass spectrometry. Even though these techniques are sensitive, they require extensive sample extraction, trained personnel, and laborious. Antibody-based assay or immunoassays method for aflatoxins and ochratoxins detection include enzyme-linked immunosorbent assay (ELISA), chemiluminescent

#### *Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

immunoassay (CLIA), fluorescence immunoassay (FIA), and lateral flow immunochromatographic assays (LFIA) [12]. Commercial ELISA kits also are widely available in the market for mycotoxins detection. However similar to chromatography analysis, ELISA method is still laboratory-bounded and requires a spectrophotometer to give accurate quantitative readings. Moreover, as ELISA is an optical-based technique, it is susceptible to light interference and hence is not suitable for on-site detection. LFIA strips also are being sought in the market as their analysis is straightforward, economical, and does not require skilled personnel. Nevertheless, the analysis is qualitative and only semiquantitative [13].

Besides immuno-based methods, spectrometric and spectroscopy techniques are also deemed sensitive for the detection of mycotoxin in food products. Near-infrared hyperspectral imaging has been successfully applied for the detection of fungal infection and OTA contamination in stored wheat and barley [14, 15]. Image processing techniques developed from machine learning is also gaining attention due to their nondestructive application for mycotoxins monitoring. Such has been applied for AFB1 in grains and feed commodities [16–19]. Machine learning methods, however, have limitations in terms of data volume, noises from the sensor, and calibration errors, besides being unable to ascertain types of mycotoxins produced.

Although tolerable limits for mycotoxins have been established, most contamination still exceeds maximum thresholds and hence continues to pose considerable risk to public health. As highlighted above, most of the reliable detections are lab-based and time-consuming. With this regard, when one requires immediate screening and on-site detection of mycotoxins, there is no readily available method. To address this, we have developed an immuno-based electrochemical biosensor for quantitative detection of *Aspergillus* spp. mycotoxins, namely AFB1 and OTA. Although the finding reported here is applied to grain corn (*Zea mays* L.), this developed biosensor platform can ultimately be applied to other food commodities.

#### **2.2 Biosensor approach for mycotoxins detection**

The first biosensor concept was introduced in 1962 based on an enzyme electrode for glucose sensing [20]. Ever since that, biosensor field has been receiving plethora of publications and vast applications in medicine [21–23], food safety [24, 25], agriculture [26–28], and environment [29]. By consensus, biosensor is defined as "*an analytical device, which exploits a biological detection or recognition system for a target molecule or macromolecule, in conjunction with a physicochemical transducer, which converts the biological recognition event into a useable output signal*" [30]. A biosensor system comprised of three essential components (**Figure 3**):


Among the transducer mechanisms, electrochemical method is favorable in comparison to optical and mechanical. While optical biosensor is susceptible to light interference, electrochemical biosensor offers feasible application with simple sample extraction, rapid detection (within a few minutes), and point-of-care measurement.

#### **Figure 3.**

*Schematic diagram of biosensor comprising three components: Detector, transducer, and output system. Taken from ref. [31].*

Main electrochemical sensing methodologies are voltammetry, potentiometry, impedimetric, conductometry, and amperometry. The recognition of biological elements (aptamer, enzyme, cell, antibody) with their targeted analytes by means of electrochemical measurement has been proven to provide reliability in numerous food contaminants detection, including mycotoxins in food and feed commodities [32–34]. Biosensors-based detection techniques have immense potential for mycotoxins detection in both research and industry as they are portable, simple, robust, sensitive, and cost-effective [35]. Of recent, aptamer, regarded as synthetic version of the antibody, also has been utilized in mycotoxin detection [36].

#### **3. Strategies for advancing mycotoxins immunosensor**

A miniaturized biosensor system with electrochemical tools and an integrated portable device for the detection of AFB1 and OTA mycotoxins is described in this study. The biosensor development employs in-house polyclonal antibody hence the term immunosensor. Research activities involved in mycotoxin immunosensor development are summarized in **Figure 4** as follows. In the first step, the antibody production is raised in rabbits as the animal host. After antibody purification and characterization, the antibody will be immobilized on screen-printed carbon electrodes (SPCEs), also addressed as sensor strips, for biosensing application. The immuno-functionalized SPCEs will be first optimized and tested on an electrochemical workstation for its proof-of-concept study. The strips are then attached to a handheld portable device integrated with I-of-Things (IoT) as point-of-care for rapid and real-time *in situ* analysis.

#### **3.1 Components in biosensor integrated internet-of-things (IoT)**

The integrated portable biosensor system for AFB1 and OTA detection with IoT system consisted of three main components (**Figure 5**): (i) an immuno-functionalized screen-printed carbon electrode (SPCE); (ii) a handheld electrochemical device with

*Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

#### **Figure 4.**

*Workflow for research activities involved in the development of immunosensor for mycotoxins detection.*

#### **Figure 5.**

*Three main components of biosensor-IoT system.* Inset*: A modified SPCE with polyaniline (PANi) and gold nanoparticle (AuNP) for antibody immobilization.*

electrochemical software; and (iii) a dedicated web server. The main part of the biosensor lies in the modified SPCEs. A plastic-based SPCE with single strip dimension of 1.3 cm (width) x 4 cm (length) is used in this study. The SPCEs have three carbonbased electrode channels: reference electrode (RE), working electrode (WE), and counter electrode (CE). WE is placed in the middle with a 4-mm circular well. The SPCEs are first modified with a conducting electroactive polymer (i.e. polyaniline, PANi) and nanogold (AuNP) network for antibody immobilization. The binding between the immobilized antibody and the targeted mycotoxin in samples will take place on the SPCE's WE surface.

Second component of the system is an improvised Android-based device with an electrochemical software and an electrode's scanner port as portable electrochemical reader. To perform the analysis, the SPCE is first inserted into the reader's port. The signal generated from antibody immobilized on the SPCE and its targeted mycotoxins is measured by the scanner on the electrochemical device *via* differential pulse voltammetry (DPV) technique. The quantified results in terms of toxin concentration (i.e. parts per billion, ppb) are displayed on the device's screen. This handheld device has built-in mobile app equipped with global positioning system (GPS) function, which allows sampling locations to be recorded. The third component, the web server, allows authorization and electrochemical settings of the portable device by registered personnel. As the portable reader is integrated with IoT, the server will collect all the field test data to allow users to perform further analysis such as location-mapping as well as trend-mapping. The system can also be set to prompt alerts to mobile phones if the test data exceeds certain preset limits to indicate immediate actions are required.

#### **4. Development of electrochemical immunosensor for mycotoxins detection**

#### **4.1 Antibody as bioreceptor**

Despite choices of aptamers and antibody as bioreceptors in mycotoxin biosensor, antibody still remains the most useful tool for identifying contaminants as the analysis can be performed on-site [6]. The production of in-house polyclonal antibody against AFB1 and OTA for mycotoxins immunosensor is described here. In biosensor development, polyclonal antibody is preferred over monoclonal antibody due to its multiple epitopes availability that can recognize its target analytes, hence signals generated are higher compared to those of monoclonal.

#### *4.1.1 Production of polyclonal antibody against mycotoxins*

Polyclonal antibodies against AFB1 and OTA were produced in-house at Biotechnology & Nanotechnology Research Centre, Malaysian Agricultural Research and Development Institute (MARDI). Antibody immunization against AFB1 and OTA was performed in four New Zealand rabbits (*Oryctolagus cuniculus*) with two replicates of rabbits for each toxin, denoted as A1 and A2 for AFB1, O1 and O2 for OTA. Animals protocol was reviewed and has been approved by the Animal Ethics Committee of MARDI (reference number 20190215/R/MAEC00045). For optimum antibody production and performance, immunization procedures were carried out adhering to the recommended guidelines [37].

Prior to immunization, blood was first taken and this preimmune batch is accounted as control study. Mycotoxins which have total molecular weight of >500 Da do not have immunogenicity property, hence as immunogen, they need to be coupled with a hapten in order to elicit immunity response in rabbits. Primary injection solution was prepared by homogenously mixing mycotoxins conjugated protein carrier with complete Freund's adjuvant (CFA) at 1:1 mixture. For subsequent immunogen, secondary injection solution was prepared by substituting CFA with incomplete Freund's Adjuvant (IFA). In total, eight immunization shots were performed and blood was collected until the fifth bleed within a duration of 5 months. Blood samples collected were purified with ammonium sulfate precipitation followed by nProtein A affinity column in order to obtain pure anti-AFB1 and anti-OTA IgG antibodies.

#### *4.1.2 Characterizations of polyclonal antibody*

Following antibody production and purification, the IgG needs to be further characterized to determine its performance, sensitivity, and selectivity. First, antibody titer is performed using indirect ELISA. The microtiters plates were coated with respective mycotoxins conjugated protein as the antigens. The performance of antibodies is evaluated by reducing the antibodies into several dilutions followed by optical measurement that is absorbance value. The purpose of titer is to determine which bleed of antibodies produced the best and most reliable result with the lowest dilution. These antibodies will then be used for further sensor development, thus, this step is exceptionally important. In general, a higher antibody titer indicates a better quality of fractionated antibody.

In both antibodies against AFB1 and OTA, preimmune antibodies that were used as control study showed no significant binding with the coated antigens (**Figures 6** and **7**), indicating the successful production of the intended antibodies. From **Figure 6**, antibodies against AFB1 from third bleed onwards showed higher absorbance compared to the preimmune and the first two bleeds. The absorbance values recorded for both A1 and A2 rabbits exhibited similar range, indicating the reliability of the polyclonal antibody production in different hosts.

For antibodies against OTA, the absorbance of all bleeds (except preimmune) showed similar pattern until 10<sup>3</sup> dilution before absorbance reading gradually decreases upon further dilution (**Figure 7**). Antibodies from second bleed onwards

**Figure 6.** *Antibody titer for anti-AFB1 antibody produced from two different rabbits, A1 and A2.*

**Figure 7.** *Antibody titer for anti-OTA antibody produced from two different rabbits, O1 and O2.*

showed the most prominent response compared to the first and preimmune bleed. Antibodies from third bleed exhibited the lowest concentration for antigen detection at 10�<sup>5</sup> antibody dilution.

The amount of total protein in each antibody batch is then quantified by protein assay. Bicinchoninic acid or BCA protein assay is usually employed to determine the concentration of protein in the developed antibody. Bovine serum albumin (BSA) is used as the reference standard for antibody against mycotoxins assay. **Figure 8** displayed the concentration of antibody protein collected from different rabbits for anti-AFB1 and anti-OTA antibodies. The calculated concentration is based on BSA standard curve. Overall, the concentration was relatively low before introduction of mycotoxin into the rabbits' immune system. The protein concentration gradually increased as the rabbits built protective immune barrier against the mycotoxins after several injections. Some rabbits maintained the amount of antibodies in their bodies and some rabbits showed low tolerance of mycotoxins after four injections.

#### *4.1.3 Cross-reaction studies*

The purified IgGs are then subjected to cross-reaction study in assessing their specificity and sensitivity. The percentage of cross-reaction (CR) is obtained based on Eq. (1) while half-maximal inhibitory concentration (IC50) values for linear response are calculated from Eq. (2). IC50 is one of the simplest and most accurate techniques to determine the performance of antibody toward specific target analyte in an assay. In this study, IC50 represents the 50% response of a series of mycotoxin concentrations (0–10 ppb) and corresponding electrochemical currents in a linear correlation, thus expressed in Eq. (2).

$$\text{CR } (\%) = \frac{IC50 \text{ } AFB1 \text{ } or \text{ } OTA}{IC50 \text{ } totimes} \text{ } \mathbf{100} \tag{1}$$

$$IC\_{50} = \frac{(0.5 - c)}{m} \tag{2}$$

**Figure 8.** *Protein concentration found in antibodies against AFB1 (left) and OTA (right) for two rabbits.*

#### *Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

The specificity of the anti-AFB1 polyclonal antibody produced was tested against other types of aflatoxins (i.e. AFB2, AFG1, and AFG2) and also other mycotoxins (ochratoxin A, ochratoxin B, fumonisin B1, fumonisin B2, and fumonisin B3). The anti-AFB1 antibody was found specific to AFB1, as indicated by the cross-reactivity studies shown in **Figure 9**. AFG1 showed cross-reaction with the antibody at 71% relative to AFB1 while AFB2 was at 65%. The cross-reaction study for AFG2 showed low cross-reactivity of less than 30%. The cross-reactivity order found was in the order of AFB1 > AFG1 > AFB2 > AFG2, corresponding with the order of toxicity reported [9, 38]. Meanwhile, when tested against other mycotoxins, anti-AFB1 antibody exhibited no cross-reaction with ochratoxins A and B, and fumonisins B1, B2, and B3. Overall, the in-house polyclonal antibody produced is highly sensitive toward AFB1, displaying high reactivity toward same group compounds and minimal crossreactivity toward different group compounds. Similarly, anti-OTA antibody produced displayed nonsignificant cross-reactivity toward all groups of mycotoxins (**Figure 9**). The performance of OTA antibody in competitive assay was highly selective toward its respective target analyte.

#### **4.2 Modified SPCEs for mycotoxins detection**

Screen-printed electrodes (SPEs) fabricated by means of thick film deposition lend themselves well in biosensor construction. The miniaturization of three-cell electrodes integrated in one chip offers an economical, straightforward analysis and practicality that suit on-site analysis application [39]. The incorporation of nanomaterials and conducting polymers on the SPE's surface has successfully enhanced the sensitivity of the detection system [40, 41]. Here, the working electrode (WE) area on the SPCE was first modified by drop-casting a mixture of PANi and AuNP for antibody immobilization.

The combination of PANi and AuNP network was found to provide numerous binding sites for biomolecules and enhance current signals due to the Au nano-size and the properties of the conducting polymer, respectively. Antibody concentration of 0.1 mg/mL, which showed the most optimum electrochemical response toward the target analyte, was immobilized on the carbon WE surface (**Figure 10a**). The

#### **Figure 9.**

*Specificity study of anti-AFB1 antibody (left) and anti-OTA antibody (right) with their targeted mycotoxins and other mycotoxins groups. The percentage of cross-reaction was calculated based on IC50 readings.*

**Figure 10.**

*(a) Working electrode (WE) of SPCE surface modification for antibody immobilization; and (b) differential pulse voltammetry (DPV measurement) for label-free approach for mycotoxin immunosensor.*

electrochemical characterization from the SPCE modification has been detailed elsewhere [42]. The label-free electrochemical biosensor approach by means of DPV measurement (**Figure 10b**) was carried out by applying a redox solution of 5 mM ferricyanide/ferrocyanide in 0.1 M KCl on the SPCEs.

#### **4.3 Sensor optimizations and standard curves development**

Processed and nonprocessed grain corns can produce harmful mycotoxins along the production chain which can lead to serious health issues for livestock and human. In grain corn, *Aspergillus* species are the main producers of mycotoxins in maize, infecting both preharvest and during storage. Standard curves for AFB1 and OTA have been developed in grain corn as sample matrix model (**Figure 11**). The electrochemical measurement was carried out in undiluted and interference-free sample matrix on an electrochemical workstation. Peak current from voltammogram for each concentration was selected as the corresponding antibody-antigen response from electrochemical process and plotted on the graph. Overall, both AFB1 and OTA displayed a good linear correlation (R2) of 0.971 and 0.9685, respectively, in a broad working range of 0–10 ppb.

**Figure 11.**

*Linear response from electrochemical workstation for a series of AFB1 (left) and OTA (right) concentrations with current reading in grain corn matrix.*

#### *Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

**Figure 12.**

*Stability study of sensor strips with immobilized antibody against mycotoxin for a duration of 48 weeks stored in different temperatures (RT and 4°C).*

#### **4.4 Storage stability of the modified SPCEs**

The stability of modified SPCEs with immobilized anti-AFB1 and anti-OTA antibodies was evaluated for the duration of 48 consecutive weeks. The sensor strips were stored without the presence of buffer at two storage temperatures: 4°C and room temperature (RT). This study is important to study the shelf-life of the functionalized strip in producing optimum results for long-term application. From **Figure 12**, it was found that storage in chilled environment showed higher antibody stability (90%) compared to ambient surroundings. The performance of the SPCEs started to decline below 90% stability after 14 weeks at 4°C and at the end of 48 weeks, the performance is still acceptable at 85% stability.

#### **5. Electrochemical portable device as point-of-care**

Portable electrochemical devices have a great potential as excellent tools for pointof-care testing technology. Detection of hazardous contaminants by means of portable electrochemical devices is a strategic approach for an efficient and rapid detection. With the complement of miniatured electrochemical cells, analysis can be conveniently performed on-site by nontechnical personnel. To facilitate on-site application and real-time detection of mycotoxins, a series of portable biosensor readers have been fabricated [39, 43, 44]. However, these readers, and even some commercially available handheld potentiostats, do not have latest features, particularly Internet-of-Things (IoT) integration. Recent trend in linking the portable device with IoT and cloud data storage has been made possible with the advancement of Fourth Industrial Revolution (IR 4.0).

In this study, an Android device is improvised as a universal portable electrochemical reader. A single unit of the portable reader is encased in a plastic box with dimensions of 16.8 cm (long) 8.5 cm (wide) 3.4 cm (height), powered by rechargeable lithium battery using USB cable through charger port. The reader which has a slot for SPCE attachment port consists of two main parts; an electrochemical transducer represented by a printed circuit board that converts signal from biology interactions reactions into readable and analyzable data; and a touch-screen Androidbased device with developed electrochemical software application (**Figure 13**). In conventional electrochemical instrument, this unit is represented by a potentiostat. A Global Positioning System (GPS) tracking app is incorporated into the portable device, which allows users to pinpoint the real-time location of the field analysis performed. The handheld device has sufficient memory size of up to 32GB that can store over 100,000 measurements.

#### **5.1 Interface for electrochemical portable reader and web server**

The device interface is designed to be user-friendly, with the aim to allow users with minimal training to perform the testing themselves in the field. Authorized users will first need to log into the system before starting the measurement. This touchscreen device features drop-down menu, which is deemed convenient and practical for users' applications. To perform the testing, the user will need to insert the biosensor strip into the SPCE port on the reader, select the type of analyte (mycotoxins) to be tested, and press 'SCAN' to start the testing process. The interface for the Androidbased device is shown in **Figure 14**. Once the scan is completed, the result is displayed on the device as concentration in ppb unit. The data then will be saved into the device, followed by the real-time transmission of the data together with GPS location and date/time to a central cloud server. The brightness of the screen can be adjusted; this feature is particularly important if the device is being used outdoor on the field. After

#### **Figure 13.**

*An android-based portable electrochemical reader unit (16.8 x 8.5 x 3.4 cm) comprised of (a) electrochemical transducer and (b) touch-screen android-based device with developed electrochemical software.*

*Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

#### **Figure 14.**

*Screen interface for portable reader application. User can select '*scan*' to begin the analysis or '*results*' to view the analyzed results. Results are displayed in form of graphs and readily calculated in concentrations (ppb).*

a successful log-in, personnel can start a measurement, review results, or change the device setting.

All analyzed results on the portable readers will be stored on the cloud server with their data log details and pinned sample location. **Figure 15** displayed the web page for IoT server that acts as authorization, electrochemical setting, and data storage platform. The website can be accessed at http://mardisense.mardi.gov.my. Complete functions of each component were described in other publications [45].

#### **5.2 Performance of portable reader as biosensing device**

#### *5.2.1 Sensor optimization on portable reader*

Antibody concentration for both AFB1 and OTA was optimized at 0.1 mg/mL and was applied for cross-reaction study using a DPV measurement. The selectivity of immobilized in-house polyclonal antibody on the SPCEs was examined through crossreactivity within the same toxins groups and other mycotoxins groups. As shown in

#### **Figure 15.**

*Login interface of MARDI*sense *website (left); and page displaying details of the analyzed data logs (right).*

**Figure 16**, the immuno-modified SPCEs were highly selective toward AFB1 and OTA, and demonstrated no cross-reaction with other mycotoxins. The cross-reactivity shown is less than 40%, hence the developed biosensor is still significantly sensitive and selective toward targeted toxins.

#### *5.2.2 Sensitivity in sample matrix*

The performance of developed sensor on the portable device was evaluated in undiluted and interference-free grain corn matrix. Standard curves for AFB1 and OTA are plotted as shown in **Figure 17**. Both AFB1 and OTA displayed an excellent linear correlation (*R*<sup>2</sup> ) of 0.9987 and 0.9836, respectively in a broad working range of 0–10 ppb. These indicate that the strategies of using the modified SPCEs on the portable reader are a success without compromising the sensitivity and accuracy of the biosensor system.

A recovery study was conducted for both AFB1 and OTA using spiked grain corn sample. This experiment is vital to evaluate the capability of the developed sensor to determine the presence of target analyte in sample. Prior to recovery study, the grain corns were first sterilized in 30% sodium hypochlorite (v/v) and autoclaved in ensuring a clean reference material for the spiking study. At concentrations of 10 ppb spiked mycotoxins, the recovery was found to be in the acceptable range of 89–96% (**Table 1**).

*Specificity study of the SPCEs with immobilized antibody with AFB1, OTA, and other mycotoxins at 6 ppb concentration.*

**Figure 17.**

*Linear response from the portable device for a series of AFB1 (left) and OTA (right) concentrations with current reading in grain corn matrix.*

*Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

#### *5.2.3 Development of immunosensing methods for mycotoxin determination in sample matrix*

The sensitivity of AFB1 and OTA detections in grain corn samples on the portable device is studied and compared with electrochemical measurements on a laboratory potentiostat workstation and an immunoassay technique that is enzyme-linked immunosorbent assay (ELISA). For the latter, a direct competitive ELISA was applied and the antibody-antigen response was recorded from the absorbance reading. All of the three detection means described here employed the in-house polyclonal antibodies against AFB1 and OTA as their bioreceptors. The laboratory potentiostat was utilized for laboratory testing while the portable device was intended for on-field testing. Standard curves for different AFB1 and OTA concentrations were developed, and parameters from the standard curves were compared, namely correlation coefficient (*R<sup>2</sup>* ), limit of detection (LOD), and limit of quantitation (LOQ) values.

From **Table 2**, all immune-based methods exhibited high *R*<sup>2</sup> values in broad working range of 0–10 ppb except for ELISA for OTA, which has a working range between 0 and 50 ppb. Electrochemical sensing method, both on the electrochemical workstation and the IoT-portable reader, displayed good correlation values, which are comparable with those of ELISA method for AFB1 and OTA. The other important parameter of determining the sensitivity of a developed method is the limit of LOD and LOQ values. LOD value represents the lowest concentration of an analyte that can be detected using an instrument. Lower LOD value implied higher sensitivity of an instrument, thus proving the better performance in detecting the target analyte. The


**Table 1.**

*Recovery study of 10 ppb AFB1 and OTA in spiked grain corn on portable-IoT device.*


**Table 2.**

*Comparison of the performance for immuno-based detection methods for AFB1 and OTA in grain corn matrix.*

MRL for both AFB1 and OTA in feed is 5 ppb and the calculated LOD values for both mycotoxins were much lower than the MRL for all immuno-based methods. In particular, the developed portable device recorded the lowest LOD and LOQ values of all for both AFB1 and OTA, signifying that the device was applicable for mycotoxins onsite testing.

#### **5.3 On-field application of portable reader**

*In-situ* determination of AFB1 was conducted at a cornfield in Perlis, northern region state of Malaysia (GPS coordinate 6.59835, 100.283) in March (pre-harvest season) and April 2022 (harvest season). Preharvest testing was carried out on 90-day-old grain corn for primary detection of AFB1 while harvest testing was carried out on 117-day-old grain corn. Samples (grain corn cob) were taken from five sampling plots, extracted using a simple sample extraction, and measured on-field (**Figure 18**). All samples showed detected AB1 concentration below the permitted level, which is 5 ppb [45].

#### **5.4 Validation study with instrumentation method**

Besides samples in Perlis, the developed biosensor system with the portable device was also tested on several other grain corn samples from Bachok and Seberang Perai, and used for AFB1 monitoring in stored grain corn with different packaging materials. A total of twenty-grain corn samples were analyzed with the portable reader and validated with HPLC-FL post-column method and UPLC-FLR. Correlation between the analyzed data and the instrumentation is tabulated in **Table 3**. LOD for the electrochemical portable reader sensor is 0.84 ppb while LOD for HPLC is 2.5 ppb. Data for HPLC method is presented by LOD reading, and it is indicative of either the AFB1 is not detected by the system or AFB1 concentration is below 2.5 ppb.

#### **Figure 18.**

*On-site application for AFB1 detection in grain corn using the modified SPCEs and portable reader.*


*Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

#### **Table 3.**

*Detection of AFBI in grain corn sample on the portable reader and its validation study using instrumentation methods (HPLC and UPLC-FLR).*

Of the 20 samples, ten samples were not contaminated by AFB1 as determined by the portable reader. Nine samples were detected with low AFB1 concentrations lower than the MRL permitted (5 ppb). Sample 15 recorded 4.42 ppb of AFB1 and was confirmed by the HPLC with 2.85 ppb. The discrepancies detection for validation with the UPLC-FLR method could be attributed to different sampling batches and duration of time for both experiments conducted, which affect the moisture content in the samples.

In general, the strategies of biosensor miniaturization for point-of-care using the modified SPCEs on the portable reader are successful without compromising the sensitivity and accuracy of the biosensor system. The portable reader offers a straightforward and direct application of AFB1 and OTA detection in grain corn with simple sample extraction as opposed to conventional methods. Furthermore, biosensor with portable reader allows the test to be performed rapidly on-site without the need to bring back the samples to the laboratory where the transportation and logistics factors may also contribute to the discrepancies in the analyzed results. The portable reader and miniaturized biosensor system described here can be used for mobile lab

applications. Although the studies presented here highlighted mainly grain corn, nevertheless the application of this developed system can be widened to other food commodities, particularly peanuts and rice for AFB1; and grains and coffee for OTA, to name a few. Moving forward, another point to be considered is the development of multi-mycotoxins detection on a dual or multiple working electrode [46].

#### **6. Conclusions**

A portable electrochemical device integrated with IoT system has been successfully designed for rapid and *in-situ* detection of *Aspergillus* spp. mycotoxins. Polyclonal antibody against AFB1 and OTA that were produced and purified in-house showed excellent sensitivity and selectivity toward the targeted mycotoxins. The antibodies are then further utilized and immobilized on modified SPCEs for the development of immunosensor system for mycotoxins detection. Using grain corn as matrix sample model, electrochemical measurements achieved by DPV for AFB1 and OTA standard curves development on the portable IoT device are superior to the ELISA method and laboratory electrochemical workstation. Good linearity was obtained in working range of 0–10 ppb (MRL 5 ppb) with excellent LOD and LOQ values. *In-situ* analysis of AB1 has been successfully conducted at a local cornfield in Perlis, northern Malaysia, using the portable device integrated with IoT as point-of-care tool. Analysis of 20 samples with the portable reader correlated well with HPLC and UPLC-FLR methods. In general, biosensors posed major advantages over the conventional method for mycotoxins detection in terms of assay duration, detection limit, and portability. With IoT integration and web server that can store data logs and pinpoint sampling locations, the portable device posed an excellent tool for mycotoxins routine monitoring in near future.

#### **Acknowledgements**

The authors would like to thank the Ministry of Agriculture and Food Industry of Malaysia for the financial support under research fund KRB 167. Gratitude also goes to other team members involved in this study and Mr. Hashim Ab Rahim from Persada Bumi Enterprise for the sampling plot at the cornfield.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Fabrication of portable device**

Electrochemical portable devices (Version 1) with cloud server integration were fabricated and developed by Biogenes Technologies Sdn. Bhd., Malaysia.

*Immunosensing of Aflatoxin B1 and Ochratoxin A on a Portable Device as Point-of-Care DOI: http://dx.doi.org/10.5772/intechopen.111724*

#### **Author details**

Nur Azura Mohd Said\*, Noor Sheryna Jusoh, Norhafniza Awaludin, Mohammad Rejab Ismail, Noor Fadilah Mohd Bakri, Lily Suhaida Mohd Sojak and Faridah Salam Biotechnology and Nanotechnology Research Centre, Malaysian Agricultural Research and Development Institute (MARDI), Selangor, Malaysia

\*Address all correspondence to: nazurams@mardi.gov.my

© 2023 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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### **Chapter 9**

## The Menace of Aflatoxin: Understanding the Effects of Contamination by *Aspergillus Species* on Crops and Human Health and Advancements in Managing These Toxic Metabolites

*Amir Afzal, Sairah Syed, Rafiq Ahmad, Muhammad Zeeshan and Ghulam Nabi*

#### **Abstract**

Food security and safety are essential global issues that require collaboration from governments, private industry, and individuals to ensure there is enough safe and nutritious food to meet the needs of a growing population. The three main elements impacting food security and safety are the availability of food, access to safe food, and the utilization of food for a healthy life. Aflatoxins, harmful mycotoxins produced by certain fungi, damage a significant proportion of the world's food supply, which is a factor in food insecurity. Effective strategies to prevent and manage aflatoxin contamination in crops include promoting sustainable and diversified agricultural practices, improving crop management, post-harvest handling and storage, and strict regulation and monitoring of food quality and safety. To date, there have been 20 different types of aflatoxins identified, with B1, B2, G1, and G2 being the most prevalent and dangerous. To mitigate the impact of aflatoxins, it is important to understand the mechanisms of contamination, the impact of aflatoxins, and the management strategies that can be employed to reduce contamination. An updated review on aflatoxin contamination, its impact and management strategies can provide valuable information for researchers, policymakers, and food safety professionals.

**Keywords:** food safety, aflatoxins, aflatoxin contamination, *Aspergillus* spp., toxic metabolites

#### **1. Introduction**

Food security and safety are indeed global issues that are becoming increasingly important as the world's population continues to grow. Food security refers to the

ability of individuals and communities to access sufficient, safe, and nutritious food to meet their dietary needs and preferences. With a growing population, it is essential to ensure that there is enough food available to meet the nutritional requirements of everyone. Food safety, on the other hand, refers to the measures taken to ensure that food is free from harmful contaminants and pathogens that could cause illness or disease. This includes everything from ensuring that food is properly stored and handled to conducting regular inspections of food processing facilities and enforcing strict food safety regulations. Ensuring both food security and food safety requires a coordinated effort from governments, private industry, and individuals. This includes investing in sustainable agriculture and food production practices, supporting local food systems, and promoting nutrition education and awareness. Ultimately, ensuring food security and safety is critical for the health and well-being of individuals and communities around the world, and it will require ongoing commitment and collaboration from all stakeholders to achieve this goal. Food security and safety are among the top priorities in today's world with a growing population. These issues are primarily influenced by three critical elements: (1) sufficient food availability, (2) accessibility to safe food, and (3) utilization of food with regard to its quality, nutritional value, and cultural significance for a healthy lifestyle. Having enough food is essential to ensure individuals have access to enough sustenance to meet their daily energy requirements and maintain good health. However, food security goes beyond mere availability and encompasses the quality and safety of the food as well. Access to safe food is also a concern, particularly for those who may not have access due to poverty, limited resources, or unavailability [1]. In some areas, food may become contaminated due to improper storage, handling, or transportation practices, making it necessary to implement measures to ensure proper food handling and storage. Finally, utilizing food in terms of quality, nutrition, and cultural significance is important for a healthy life. Food provides necessary nutrients for growth and body maintenance, and also has a significant cultural and social impact [2]. A balanced and varied diet is necessary to maintain good health and prevent chronic diseases. In conclusion, addressing these crucial elements of food security and safety is imperative for the overall well-being of the global population. If any of these components fail, it results in food insecurity and malnutrition that has a detrimental impact on human health and the social and economic well-being of society. Furthermore, contamination of food and feed by mycotoxins is a significant contributor to the problem of food insecurity [3]. Aflatoxins are a type of mycotoxin primarily produced by the fungi *Aspergillus flavus* and *A. parasiticus* and have been extensively studied [4]. In addition to the human and animal health risks associated with aflatoxin contamination, it can also have significant economic impacts. Aflatoxin-contaminated crops may be rejected by food processors and retailers, resulting in reduced market value and financial losses for farmers. In some cases, crops may need to be destroyed entirely, which can result in even greater economic losses. The contamination of a diverse range of foods and feeds with AF can lead to economic losses, and various factors such as season, post-harvest and management practices, food type, and geographic location can contribute to this contamination [5]. This review serves as a valuable resource for researchers as it provides crucial information that can be used to devise effective mitigating strategies.

According to the Centers for Disease Control and Prevention (CDC), mycotoxin exposure is a chronic issue affecting an estimated 4.5 billion people [6]. However, a recent study suggests that the global occurrence of mycotoxin contamination in crops is between 60 and 80% [7].

*The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

#### **2. Aflatoxin-producing species of** *Aspergillus* **and their diversity**

Aflatoxins, which are primarily produced by *Aspergillus flavus* and A. parasiticus, are the mycotoxins known to be produced by species of *Aspergillus*. However, other species such as *A. nomius, A. pseudotamarii, A. parvisclerotigenus,* and *A. bombycis* from section *Flavi*, *A*. *ochraceoroseus* and *A. rambellii* from section *Ochraceorosei*, as well as *Emericella astellata* and *Epipleoneura venezuelensis* from the *Nidulatans*, have also been identified as producers of aflatoxins [8]. There are multiple types of aflatoxins that have been documented, and their presence in crops and food is a major global concern, particularly for crops of economic significance (**Figure 1**). Aflatoxins are toxic compounds produced by certain species of fungi, including *Aspergillus flavus* and *A. parasiticus* [10].

The genus *Aspergillus* is comprised of four subgenera and a total of 339 species [11]. The *Aspergillus* genus is a diverse group of fungi, which includes over 200 species [12]. The agriculturally important species of *Aspergillus* that produce aflatoxin belong to the *Flavi* section [13]. *A. flavus* and *A. parasiticus* are known to contaminate a variety of crops including maize, peanuts, cottonseed, tree nuts, and spices, among others [14]. There are two different types of *A. flavus*, a fungus that creates aflatoxins, based on the size of their sclerotia: L and S morphotypes. The L morphotype produces many spores and a range of aflatoxin levels, but only a few large sclerotia (>400 μm). The S morphotype produces fewer spores, but consistently high levels of aflatoxins, and many small sclerotia (<400 μm) [15].

#### **3. Classification and types of aflatoxins**

The names of these aflatoxins are derived from their property of absorbing and emitting light at distinct wavelengths. Aflatoxins B1 and B2 fluoresce blue when subjected to ultraviolet light with a wavelength of 425 nm, while aflatoxins G1 and G2 fluoresce green when exposed to ultraviolet light with a wavelength of 540 nm [5]. Aflatoxins are difuranocoumarins that can be divided into two categories based on their chemical structure: the difurocoumarocyclopentenone series, which includes AFB1, AFB2, AFB2A, AFM1, AFM2, AFM2A, and aflatoxicol; and the difurocoumarolactone series, which includes AFG1, AFG2, AFG2A, AFGM1, AFGM2, AFGM2A, and

AFB3 [16]. More than 20 distinct types of aflatoxins have been identified to date [17]. However, the most commonly and widely known aflatoxins are aflatoxins B1, B2, G1, G2, M1, M2, aflatoxicol, and aflatoxin Q1 [18].

#### **4. Environmental conditions favoring aflatoxin production on crops**

The growth of these fungi is commonly observed in warm and humid conditions, which are prevalent in tropical and subtropical regions [19]. They are commonly found on crops such as corn, peanuts, cottonseed, and tree nuts, which are stored in warm and humid conditions for extended periods of time. This provides an ideal environment for the growth and spread of aflatoxin-producing fungi, leading to contamination of these crops and the potential health hazards and economic losses that come with it [20].

#### **5. The carcinogenic properties of aflatoxins B1, B2, G1, and G2**

Of all the types of aflatoxins, aflatoxins B1, B2, G1, and G2 have received the most extensive research attention and are the most commonly found, and they have been shown to have the most significant impact on human and animal health [21, 22]. These are considered to be more important due to their widespread prevalence in food [5]. Aflatoxins B1, B2, G1, and G2 are four major types of aflatoxins that are classified as Group 1 carcinogens. Among them, the most toxic and commonly found in crops is aflatoxin B1 (AFB1) [23]. Moreover, aflatoxin B1 can bind to DNA and modify its structure, resulting in genotoxic effects [24]. Different species of *Aspergillus* fungi produce different types of aflatoxins. Aflatoxins B1, B2, G1, and G2 are produced by *Aspergillus bombycis*, *A. nomius*, *A. parasiticus*, *A. parvisclerotigenus*, *A. pseudocaelatus*, *A. minisclerotigenes*, and *A. arachidicola*. However, species such as *A. flavus*, *A. ochraceoroseus*, and *A. rambellii* only produce aflatoxin B1 and B2, while others such as *Aspergillus pseudonomius, A. pseudotamarii, Emericella astellata*, *E. olivicola*, and *Epipleoneura venezuelensis* produce only aflatoxin B1 [25]. The occurrence of aflatoxins was observed in the following decreasing order: AFG2 > AFG1 > AFB2 > AFB1, where: AFG1: Aflatoxin G1, AFG2: Aflatoxin G2, AFB1: Aflatoxin B1, AFB2: Aflatoxin B2. Note that the ">"sign indicates "greater than" and is used to show the decreasing order of occurrence, with AFG2 being the most prevalent and AFB1 being the least prevalent with concentration ranges of 0.78 ± 0.04–234.73 ± 3.8 μg/kg, 0.47 ± 0.03– 21.6 ± 0.33 μg/kg, 1.01 ± 0.05–13.75 ± 1.2 μg/kg, and 0.66 ± 0.06–5.51 ± 0.26 μg/ kg, respectively. Of the 100 samples analyzed for total aflatoxins (total AFs), 68 (68%) exceeded the limits set by the EC, with concentration ranges of 4.98 ± 0.6– 445.01 ± 8.9 μg/kg. Similarly, 58 (58%) of the samples exceeded the limits set by GSA, with concentration ranges of 12.12 ± 1.4–445.01 ± 8.9 μg/kg [26].

#### **6. Understanding the health effects of aflatoxin exposure**

Aflatoxin contamination is a significant public health concern, particularly in developing countries where food safety regulations may be less stringent and where poverty and malnutrition can exacerbate the health effects of exposure [27]. The European Commission and the U.S. Food and Drug Administration have set a

*The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

maximum limit of 20 ppb (parts per billion) of aflatoxins in food and feed products for human and animal consumption to help prevent these health hazards and economic losses. Aflatoxins are both carcinogenic and mutagenic in nature and can result in aflatoxicosis in humans and animals [5]. Recently, a form of pulmonary aspergillosis has been linked to the coronavirus disease (COVID-19). It has been established that patients with compromised immune systems are more likely to experience severe cases of COVID-19 when complicated by pulmonary aspergillosis. To date, there have been 20 reported cases of COVID-19 associated pulmonary aspergillosis (CAPA) worldwide [28]. Recently, a correlation between the coronavirus disease (COVID-19) and pulmonary aspergillosis has also been reported, with 20 cases of coronavirus disease-associated pulmonary aspergillosis (CAPA) recorded globally. It is noted that the severity of COVID-19 symptoms is increased in immunocompromised patients with pulmonary aspergillosis [29]. Given their widespread presence, around 4.5 billion people worldwide are estimated to be exposed to aflatoxin contamination [30]. The adverse effects of aflatoxins on living organisms have led to the setting of a maximum limit of 20 ppb (parts per billion) for aflatoxins in food and feed products intended for human and animal consumption by both the European Commission and the U.S. Food and Drug Administration [31]. The adverse health effects of aflatoxin contamination can include acute toxicity, immune suppression, liver damage, and an increased risk of developing liver cancer. Aflatoxin contamination can have a range of adverse health effects in both humans and animals, and these effects can vary depending on the level and duration of exposure. The most significant health effects of aflatoxin contamination include the following.

#### **6.1 The adverse health effects of aflatoxin contamination and the importance of prevention and control measures**

High levels of aflatoxin exposure can cause acute toxicity, which can result in a range of symptoms, such as vomiting, abdominal pain, convulsions, coma, and even death ([32] Available from: https://www.ncbi.nlm.nih.gov/books/NBK557781/). These symptoms occur due to the liver's inability to detoxify aflatoxins effectively, leading to a buildup of toxic metabolites in the body. When consumed, aflatoxins are primarily metabolized in the liver, where they can cause damage to liver cells and impair liver function. The liver is responsible for breaking down and eliminating many toxins, including aflatoxins, from the body. However, in cases of high-level exposure, the liver may not be able to metabolize and eliminate the aflatoxins effectively, leading to a buildup of toxic metabolites in the body [33]. The severity of the symptoms of acute toxicity depends on several factors, including the level and duration of aflatoxin exposure, as well as the individual's age, nutritional status, and overall health. Young children and people with weakened immune systems or liver disease are particularly vulnerable to the adverse effects of aflatoxin exposure, as their bodies may not be able to detoxify the aflatoxins effectively. In addition, malnutrition can exacerbate the health effects of aflatoxin exposure, as it can weaken the body's ability to fight off infections and other health problems. This can lead to an increased risk of complications and death in cases of acute aflatoxin toxicity. Overall, the adverse health effects of aflatoxin contamination, including acute toxicity, can have significant impacts on public health and food safety, particularly in developing countries where food safety regulations may be less stringent, and where poverty and malnutrition can exacerbate the health effects of exposure. Implementing appropriate prevention and control measures, regular monitoring and testing, and appropriate

food safety regulations, as well as promoting public awareness and education about the risks of aflatoxin exposure, are critical to preventing the adverse health effects of aflatoxin contamination.

#### **6.2 Aflatoxin exposure and the increased risk of infections and diseases**

Aflatoxins are known to have immunosuppressive effects, which means that they can impair the body's immune system and its ability to fight off infections and other diseases. A weakened immune system can increase the risk of various health problems, including bacterial and viral infections, as well as chronic illnesses such as cancer. The immunosuppressive effects of aflatoxins can occur through various mechanisms, including the disruption of immune cell function and the impairment of cytokine production. Aflatoxins have been shown to reduce the production of cytokines, which are proteins that regulate the immune response and play a critical role in fighting infections and diseases. In addition, aflatoxins can damage the liver, which can impair its ability to produce proteins that are important for immune function. The immunosuppressive effects of aflatoxins are particularly concerning for individuals who are already immunocompromised, such as those with HIV/AIDS, cancer, or other chronic illnesses. For these individuals, exposure to aflatoxins can further weaken their immune system, making them more susceptible to infections and other health problems. Coulombe [34] described the various routes of exposure to mycotoxins, such as ingestion, inhalation, and dermal contact. He discussed the factors that can influence the toxic effects of mycotoxins, such as dose, duration of exposure, and the age and health status of the individual.

#### **6.3 Aflatoxins as carcinogens: understanding the link to liver cancer**

Aflatoxins are toxic compounds that are primarily metabolized by the liver. When ingested, they are absorbed from the gastrointestinal tract and transported to the liver, where they are metabolized into a range of byproducts, some of which are highly reactive and can cause damage to liver cells. The liver plays a critical role in the detoxification of aflatoxins and other harmful substances, but when exposed to high levels of aflatoxins, the liver can become overwhelmed, and its ability to metabolize and eliminate the toxins can be impaired. This can lead to the accumulation of aflatoxin metabolites in the liver and other tissues, which can cause liver damage and even liver failure. Liver damage caused by aflatoxin exposure can manifest in various ways, including acute and chronic forms. Acute liver damage caused by aflatoxin exposure is characterized by rapid onset and is associated with symptoms such as jaundice, abdominal pain, and liver failure. Chronic liver damage caused by aflatoxin exposure, on the other hand, is associated with long-term exposure to lower levels of the toxin and can result in the development of liver cirrhosis or liver cancer. Individuals who are at higher risk of liver damage caused by aflatoxin exposure include those who consume a diet high in aflatoxin-contaminated foods, as well as those who have pre-existing liver disease or other risk factors that compromise liver function [35].

HCC is a primary liver cancer that arises from the hepatocytes, which are the main functional cells of the liver. HCC is a major global health problem, and its incidence has been increasing in many countries worldwide. The risk factors for HCC, which include chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohol consumption, obesity, and exposure to aflatoxin. The prevalence of these risk factors varies in different regions of the world and among different populations [36].

#### *The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

Chronic exposure to aflatoxins has been shown to increase the risk of developing liver cancer, a type of cancer that affects the liver [30]. Aflatoxins are naturally occurring toxins produced by certain molds, primarily *Aspergillus flavus* and *Aspergillus parasiticus*, which can contaminate food and feed crops, particularly those that are stored in warm and humid conditions. When contaminated crops are consumed by humans or animals, aflatoxins can enter the bloodstream and be transported to the liver, where they can cause DNA mutations and other cellular damage that can lead to the development of cancerous cells.

Aflatoxins are classified as Group 1 carcinogens by the International Agency for Research on Cancer (IARC), which means that there is sufficient evidence to suggest that they are carcinogenic to humans. Aflatoxin exposure is a significant risk factor for liver cancer in areas where food and feed contamination are common, such as sub-Saharan Africa, Southeast Asia, and parts of South America. The liver is the primary organ responsible for detoxifying aflatoxins, but chronic exposure to high levels of aflatoxins can overwhelm the liver's capacity to metabolize and eliminate the toxins, leading to the accumulation of DNA-damaging metabolites and increased risk of liver cancer [37]. The risk of developing liver cancer from aflatoxin exposure is further increased in individuals with pre-existing liver disease, such as viral hepatitis B or C, as well as those who consume alcohol or have a weakened immune system. Aflatoxins are highly toxic and carcinogenic to both humans and animals, and exposure to these toxins can lead to liver damage, immune system suppression, and even death in severe cases. As a result, there are ongoing global efforts to better understand the genetics, biochemistry, and regulation of aflatoxin biosynthesis, as well as the taxonomy, biology, toxicology, and evolution of aflatoxigenic fungi.

Some of the key areas of research in this field include identifying the genes and pathways involved in aflatoxin biosynthesis [38], developing new methods for detecting and quantifying these toxins, and exploring potential strategies for preventing or reducing aflatoxin contamination in crops. By gaining a better understanding of these factors, researchers hope to develop new tools and approaches for mitigating the risk of aflatoxin exposure and protecting public health.

#### **7. Understanding the genetics and biochemistry of aflatoxin biosynthesis: implications for food safety and public health**

Identifying the genes and pathways involved in aflatoxin biosynthesis is an important area of research for several reasons. First, it can help us to better understand the mechanisms by which aflatoxins are produced by fungi, which can in turn inform strategies for preventing or reducing their production. Second, it can help us to develop new tools for detecting and quantifying aflatoxins in crops and food products, which is essential for ensuring food safety. Over the years, researchers have made significant progress in identifying the genes and pathways involved in aflatoxin biosynthesis. This work has been facilitated by advances in genomics and bioinformatics, which have enabled researchers to sequence and analyze the genomes of aflatoxin-producing fungi. Some of the key genes and pathways involved in aflatoxin biosynthesis include the following:

1.The aflatoxin biosynthetic gene cluster (AF cluster): This cluster contains around 25 genes that are involved in the biosynthesis of different intermediates in the aflatoxin pathway [39]. The cluster is regulated by a complex network of

transcription factors that respond to environmental signals, such as temperature [40], pH [41], and nutrient availability [42, 43].


By studying the function of these genes and their products, researchers can gain a deeper understanding of how aflatoxins are produced and regulated in fungi, and develop new approaches for preventing or reducing their production. Payne & Brown [47] highlights the importance of understanding the genetic and physiological mechanisms of aflatoxin biosynthesis in developing effective strategies for controlling its production and minimizing its impact on human and animal health.

#### **8. Advances in methods for detecting and quantifying aflatoxins in crops and food products: implications for food safety**

Monitoring and controlling aflatoxin contamination in food products is crucial to ensure food safety and protect public health. By establishing reliable testing methods and implementing prevention strategies, we can minimize the risk of exposure to aflatoxins and mitigate their adverse health effects [48]. Developing new methods for detecting and quantifying aflatoxins is essential for ensuring food safety and protecting public health. Traditional methods for detecting aflatoxins include thin-layer chromatography (TLC) [49] and high-performance liquid chromatography (HPLC), which are effective but time-consuming and labor-intensive [50]. In recent years, several new methods have been developed for detecting and quantifying aflatoxins in crops and food products. Some of these methods include the following:

#### **8.1 Immunoassays**

Immunoassays are based on the use of antibodies that specifically recognize and bind to aflatoxins. Immunoassays can be performed using a range of formats [51], including ELISA (enzyme-linked immunosorbent assay) [52, 53], lateral flow tests [54], and fluorescent assays [55].

*The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

#### **8.2 Mass spectrometry**

Mass spectrometry is a powerful analytical technique that can detect and quantify the presence of aflatoxins with high sensitivity and specificity. Mass spectrometry can be coupled with different separation techniques, such as liquid chromatography (LC) or gas chromatography (GC), to achieve high levels of separation and detection [56].

#### **8.3 Biosensors**

Biosensors are devices that use biological components, such as enzymes or antibodies, to detect and quantify the presence of aflatoxins in food samples [57]. Biosensors can be based on different transduction principles, such as electrochemical, optical, or piezoelectric transduction [58].

#### **8.4 DNA-based methods**

DNA-based methods use DNA probes or PCR (polymerase chain reaction) to detect the presence of aflatoxin-producing fungi in crops or food products. DNAbased methods can be faster and more sensitive than traditional methods, but they require specialized equipment and expertise [59].

By developing new methods for detecting and quantifying aflatoxins, researchers can improve our ability to monitor and control the presence of these toxins in food products, and ensure that they meet regulatory standards for food safety.

#### **9. Aflatoxin prevention for food safety**

Exploring potential strategies for preventing or reducing aflatoxin contamination in crops is critical for ensuring food safety and protecting public health. Some of the key strategies that researchers are investigating in this area include the following:

#### **9.1 Breeding for resistance against aflatoxin contamination**

Breeding groundnut varieties with stable resistance to aflatoxin contamination is a sustainable and effective approach to reducing the problem. However, this poses challenges to breeders due to the limited availability of improved germplasm and the significant genotype-by-environment (GxE) interaction for aflatoxin contamination. The limited germplasm restricts the range of genetic variability that breeders can work with, making it difficult to identify suitable parental lines for breeding. Additionally, the significant GxE interaction means that the performance of a genotype in one environment cannot be used to predict its performance in another environment, making it hard to select for resistance across diverse environments. Overcoming these challenges will require breeders to utilize innovative breeding techniques, such as marker-assisted selection, genomic selection, and multi-environment testing, to identify and incorporate favorable alleles for resistance across diverse environments [60, 61]. To combat this problem, researchers have been working on developing crops that are resistant to aflatoxin contamination. Aflatoxin biosynthesis in *Aspergillus* spp. is regulated by oxidative stress responses, which are induced by environmental stresses such as drought and heat stress. Host-derived reactive oxygen species (ROS) may play a role in cross-kingdom communication between host plants and *A. flavus*.

#### *9.1.1 Application of RNA interference (RNAi) technology*

Recent advances in plant breeding technology have enabled the study and application of metabolomic, proteomic, and transcriptomic knowledge in productive breeding populations [62]. Researchers are exploring ways to engineer crops that are resistant to aflatoxin-producing fungi or that produce antifungal compounds that can inhibit the growth of these fungi. However, there have been some promising advancements in engineering aflatoxin-resistant crops. One approach is to use RNA interference (RNAi) technology to silence genes in the fungi responsible for aflatoxin production [63, 64]. Another approach is to introduce genes from other organisms, such as bacteria or plants, that can break down aflatoxins [65]. Breeders now have more options for their improvement programs as there are a greater number of maize breeding lines that demonstrate resistance to both *A. flavus* infection and aflatoxin accumulation. The majority of these resistant lines have a tropical background, but newer lines have been created through crosses and backcrosses between tropical and temperate germplasm [66]. The GEM project has been instrumental in developing many of these lines through breeding crosses and hybrids [67]. Genomic regions that offer a consistent increase in resistance to aflatoxin or *A. flavus* induced ear rot in resistant maize lines have been identified through various studies, including several QTL and one meta-QTL analysis. Each of these QTL typically accounts for 5–20% of the observed variation in resistance. However, it is possible that the effects of some of these QTL may have been overestimated due to the Beavis effect [68].

#### *9.1.2 The introduction of genes from other organisms*

Another approach is to use genetic engineering to introduce genes that can enhance plant defense mechanisms against fungi, such as the expression of antifungal proteins or enzymes [69]. For example, researchers have successfully introduced genes from a wild peanut species into commercial peanut cultivars to increase their resistance to aflatoxin-producing fungi [70]. Overall, while there are still many challenges to overcome in developing aflatoxin-resistant crops, there is promising progress being made. The development of such crops could have significant benefits in improving food safety, reducing health risks, and increasing food security, especially in developing countries where aflatoxin contamination is a major problem.

#### **10. Crop management practices**

Aflatoxin contamination is strongly influenced by environmental factors, such as temperature, humidity, and rainfall, as well as cultural practices, such as planting density, irrigation, and fertilization. Researchers are exploring ways to optimize these factors to reduce the risk of aflatoxin contamination in crops. To prevent the adverse health effects of aflatoxin contamination, it is important to implement appropriate prevention and control measures.

#### **10.1 Preharvest amendments**

Preharvest amendments are treatments or additives applied to crops or soil before harvest in order to improve crop quality, yield, or post-harvest performance. The use of preharvest amendments is common in agricultural practices, as they can help to address *The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

a range of issues affecting crop production and quality. For example, preharvest amendments may be used to control pests and diseases, improve soil fertility and nutrient availability, enhance plant growth and development, or reduce post-harvest losses and spoilage. However, it is important to carefully consider the potential risks and benefits associated with the use of preharvest amendments, as some may have negative impacts on the environment or human health. In addition, regulations governing the use of preharvest amendments may vary depending on the crop, location, and other factors.

#### *10.1.1 The role of high-quality seeds in preventing aflatoxin contamination in agriculture*

Using high-quality seeds is an important part of preventing aflatoxin contamination in food production. High-quality seeds are typically produced using good agricultural practices, such as careful selection and handling of parent plants, testing for disease and pests, and appropriate storage and transport conditions. By using high-quality seeds, farmers can ensure that their crops are healthy and better able to resist fungal infections that can lead to aflatoxin contamination. This is because healthy plants are better able to defend themselves against pathogens and are less likely to develop the kinds of stress conditions that can make them more susceptible to fungal infections.

#### *10.1.2 Managing aflatoxin contamination in groundnuts through soil amendments*

Ijaz et al. [71] proposed the use of soil amendments has been as a potential strategy for managing aflatoxin contamination in groundnuts. Several studies have investigated the effects of different soil amendments on aflatoxin levels in groundnuts, including organic amendments such as poultry manure, vermicompost, and green manure, as well as Inorganic amendments such as lime and sulfur. Overall, the results of these studies have been mixed, with some studies reporting significant reductions in aflatoxin levels following the application of soil amendments, while others have found little to no effect. The effectiveness of soil amendments in reducing aflatoxin levels may depend on a variety of factors, including the type and amount of amendment used, the timing and frequency of application, and the local environmental conditions. Despite the inconsistent results, the use of soil amendments may still be a promising approach for managing aflatoxin contamination in groundnuts, particularly in areas where other strategies, such as chemical fungicides, are not available or feasible. However, more research is needed to better understand the mechanisms underlying the effects of soil amendments on aflatoxin contamination, as well as to optimize their use in different agricultural settings.

#### *10.1.3 The importance of crop rotation in preventing aflatoxin contamination*

Proper crop rotation is an important practice that can help reduce the risk of aflatoxin contamination in agriculture. This is because crop rotation helps to interrupt the life cycle of the fungi that produce aflatoxins, which can help prevent the buildup of fungal spores and reduce the risk of contamination in subsequent crops. Crop rotation involves planting different crops in a field over successive growing seasons. This helps to break the cycle of plant-specific pests and diseases, which can build up in the soil over time and infect subsequent crops. By alternating crops, farmers can help to disrupt the life cycle of these pests and diseases, which can help to reduce their populations and limit their impact on subsequent crops. In the case of aflatoxin contamination, crop

rotation can help to reduce the buildup of fungal spores in the soil. This is because the fungi that produce aflatoxins typically infect specific crops, such as corn and peanuts. By rotating these crops with other types of crops, such as legumes, cereals, or grasses, farmers can help to reduce the buildup of fungal spores in the soil, which can in turn reduce the risk of contamination in subsequent crops [72–74].

#### *10.1.4 Preventing aflatoxin contamination through effective Pest control measures*

Aflatoxigenic fungi can grow on crops both in the field and during storage, and their growth is often facilitated by the presence of pests and insects. Pests such as insects and rodents can damage crops, creating entry points for fungi that produce aflatoxins. Proper pest control measures can help prevent crop damage and reduce the risk of fungal infections. Effective pest control measures can include the use of chemical or biological pesticides, crop sanitation practices, and appropriate storage and transport practices. Integrated pest management (IPM) is a holistic approach to pest control that uses a combination of techniques to minimize the use of pesticides while still effectively managing pests. IPM can be an effective approach to preventing aflatoxin contamination while also minimizing the use of potentially harmful chemicals in food production [75].

#### *10.1.5 Biological control of aflatoxins*

#### *10.1.5.1 Using non-toxigenic strains of* Aspergillus *spp.*

Biological control of aflatoxin using non-toxigenic strains of *Aspergillus* spp. has shown promising results in both laboratory and field studies. These non-toxigenic strains can outcompete and displace the toxigenic strains, reducing the overall levels of aflatoxin contamination in crops [3]. The use of non-toxigenic strains for biological control of aflatoxin is considered to be a safe and environmentally friendly approach. These strains are naturally occurring and do not produce harmful toxins, making them ideal for use in agricultural settings [76]. Several non-toxigenic strains of *Aspergillus* spp. have been identified and tested for their ability to control aflatoxin contamination in crops, including *Aspergillus flavus* AF36 and *Aspergillus parasiticus* NRRL 2999. Studies have shown that these strains can significantly reduce the levels of aflatoxin contamination in crops, including maize, peanuts, and tree nuts. PCR assays can be used to distinguish between genetically similar toxigenic and atoxigenic isolates of *A. flavus* [77]. Specifically, atoxigenic isolates with deletions within the aflatoxin gene cluster can be readily identified using this method. While the use of non-toxigenic strains for biological control of aflatoxin is still in the early stages of development, it shows great potential as a strategy for reducing aflatoxin contamination in crops and improving food safety. Further research is needed to better understand the effectiveness of this approach in different agricultural settings and to develop practical methods for implementing it on a large scale [78].

#### *10.1.5.2 Effectiveness of Trichoderma species and cattle dung as soil amendment in reducing aflatoxin contamination in groundnut*

A study conducted recently aimed to investigate the effect of using *Trichoderma* species in combination with cattle dung as a soil amendment on the yield and preharvest aflatoxin contamination of groundnut. The researchers found that the application of *Trichoderma* species, in combination with cattle dung, significantly improved

*The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

the yield of groundnut and reduced preharvest aflatoxin contamination. Moreover, the study also showed that the application of *Trichoderma* species and cattle dung improved soil fertility and increased the availability of nutrients in the soil. The study suggests that the use of *Trichoderma* species in combination with cattle dung can be an effective strategy to improve crop productivity and reduce aflatoxin contamination in groundnut cultivation [79].

#### **10.2 Post-harvest management**

Post-harvest management plays a crucial role in reducing aflatoxin contamination in crops. Aflatoxins are a group of mycotoxins produced by the fungus *Aspergillus flavus* and *Aspergillus parasiticus* that can contaminate crops during preharvest, harvest, and post-harvest stages. Here are some post-harvest management practices that can help reduce aflatoxin contamination:

#### *10.2.1 Mitigating aflatoxin contamination through appropriate storage and drying conditions*

Proper storage and drying conditions are critical to mitigating the risk of aflatoxin contamination in food production. After harvest, crops should be stored in dry, cool, and well-ventilated facilities to prevent the growth of fungi that can produce aflatoxins. Crops dried to the appropriate moisture content prevent fungal growth and minimize the risk of contamination. Proper drying conditions may vary depending on the type of crop and the local climate, but generally involve careful monitoring of temperature, humidity, and airflow. Drying can be done using a variety of methods, such as natural drying in the sun or with fans and heaters, or using specialized equipment such as dryers and dehumidifiers. Proper storage and drying practices can significantly reduce the risk of aflatoxin contamination in food production and help ensure the safety and quality of food products [80]. Mousavi Khaneghah et al., [81] emphasizes the importance of understanding and addressing the risks associated with aflatoxin contamination in cereals. While the production of these toxins is a natural occurrence, there are several factors that can contribute to increased contamination, such as improper storage and handling practices.

#### **11. Conclusion**

Food security is crucial for human health and socioeconomic stability, but mycotoxins produced by fungi can contaminate crops, leading to health problems and economic losses. Aflatoxin is a mycotoxin that commonly contaminates crops such as corn, peanuts, and cottonseed, and can cause serious health consequences. Preventing and managing aflatoxin contamination requires effective strategies such as improved crop management practices, post-harvest handling and storage, and strict regulation and monitoring of food quality and safety. There are several strategies that researchers are exploring to prevent or reduce aflatoxin contamination in crops. Some of these strategies include the following:

**Good agricultural practices:** One of the primary ways to prevent aflatoxin contamination is to implement good agricultural practices, such as crop rotation, proper irrigation, and the use of high-quality seeds. These practices can help minimize the growth of *Aspergillus* fungi, which are responsible for producing aflatoxins.

**Biological control:** Researchers are also investigating the use of biological control agents, such as non-aflatoxigenic strains of *Aspergillus* fungi or other microorganisms, to reduce the growth of aflatoxin-producing fungi. This strategy involves introducing these microorganisms into the soil or onto crops to compete with and displace the aflatoxin-producing strains.

**Chemical control:** Chemical control methods, such as the use of fungicides or insecticides, can also be effective in preventing aflatoxin contamination. However, these methods can be costly and may have negative environmental impacts.

**Post-harvest interventions:** Another strategy for reducing aflatoxin contamination is to implement post-harvest interventions, such as proper drying and storage techniques, to prevent the growth of *Aspergillus* fungi and minimize aflatoxin production.

**Genetic modification:** Researchers are also investigating the use of genetic modification to develop crops that are more resistant to aflatoxin contamination. This strategy involves modifying the genetic makeup of crops to enhance their ability to resist *Aspergillus* fungi and reduce the production of aflatoxins. Breeding groundnut varieties with stable resistance to aflatoxin contamination is a sustainable approach but poses challenges due to limited germplasm and significant genotype-by-environment interaction. Overcoming these challenges will require breeders to utilize innovative breeding techniques and strategies, through collaborative efforts among breeders, geneticists, and agronomists.

In summary, preventing the adverse health effects of aflatoxin contamination requires a multi-pronged approach that involves implementing appropriate prevention and control measures, regular monitoring and testing, and appropriate food safety regulations, as well as promoting public awareness and education about the risks of aflatoxin exposure. In addition to safeguarding the health of consumers, such practices can enhance the quality and market value of their produce. Overall, a combination of these strategies may be necessary to effectively prevent or reduce aflatoxin contamination in crops and ensure food safety.

#### **Conflict of interest**

None.

*The Menace of Aflatoxin: Understanding the Effects of Contamination by* Aspergillus Species… *DOI: http://dx.doi.org/10.5772/intechopen.110782*

#### **Author details**

Amir Afzal1 \*, Sairah Syed1 , Rafiq Ahmad1 , Muhammad Zeeshan<sup>2</sup> and Ghulam Nabi3

1 Barani Agricultural Research Institute, Chakwal, Pakistan

2 Barani Agricultural Research Station, Fateh Jang, Pakistan

3 Groundnut Research Station, Attock, Pakistan

\*Address all correspondence to: rajaamirafzal@gmail.com

© 2023 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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#### **Chapter 10**

## Whey Protein Fermentation with *Aspergillus niger*: Source of Antioxidant Peptides

*Marcela Patricia Gomez Rojas and Oscar Marino Mosquera Martinez*

#### **Abstract**

*Aspergillus niger* is a filamentous fungus that through its proteolytic activity, as a result of its proteases, hydrolyzes whey proteins into smaller peptides. These peptides are characterized by antioxidant properties due to the presence of specific amino acids, such as histidine, tyrosine, tryptophan, cysteine, and methionine, which have been shown to have antioxidant effects. Considering the above, peptide extracts derived from the fermentation of a lactic serum substrate with *Aspergillus niger* were obtained, which were partially purified by precipitation with ZnSO4/acetone; subsequently, the antioxidant capacity was evaluated by spectrophotometric techniques as 2,2-azinobis-3ethyl benzothiazole-6-sulfonic acid (ABTS▪<sup>+</sup> ), diphenylpicrylhydrazyl (DPPH▪ ), in 96-well microplates, these analyses showed that these extracts have an antioxidant activity higher than 50%; likewise, the amount of thiol groups (-SH) was determined to be higher than 29 nmol/μL and the superoxide dismutase activity (SOD) with values above 0.010 SOD units/mL. For this reason, it is proposed that they can be studied in the future as substances within a food supplementation or in the therapeutic field.

**Keywords:** *Aspergillus niger*, proteolytic activity, antioxidant activity, fermentation, whey

#### **1. Introduction**

Peptides are low molecular weight protein fragments, consisting of 2 to 20 amino acid residues [1–3], some of which exhibit physiological effects, beneficial to humans, which is why they are called bioactive peptides [4]. These are obtained mainly by hydrolysis of precursor proteins during fermentation processes with exogenous enzymes [5] from plant, bacterial or fungal sources [6], or through their expression and secretion during metabolic processes [7].

Fungal sources have aroused special interest because they exhibit a number of exogenous enzymes with applicability at pharmacological and industrial level [8]; among the most widely used fungal species is the genus *Aspergillus*, whose species *Aspergillus oryzae* [9], *Aspergillus flavipes* and *Aspergillus niger* [10], when used as an enzymatic source gave way to obtain peptides with antihypertensive [11], antioxidant [12], antidiabetic [13], antimicrobial [9], and antioxidant [14] bioactivities, among others.

Likewise, among the sources of precursor proteins used as substrates by fungal enzymatic sources are various kinds of milk and their derivatives [15], gelatin [11], grains such as lentils [13] and soy [16], and even eggs [17]. It should be noted that of these protein sources, milk is the most consumed worldwide [18] and its proteins have different biological and nutritional properties [19], and make it a source of bioactive peptides, which are released from precursor proteins, such as α-lactalbumin (α-LA), β-lactoglobulin (β-LG), caseins (CN), immunoglobulins (Ig), lactoferrin (LF), peptide-protein fractions, phosphoglycoproteins and minor serum proteins (transferrin and serum albumin) [20], during processes already mentioned such as fermentation, chemical hydrolysis or enzymatic hydrolysis [21].

Products derived from fermentation processes have been relevant in people's diets because their nutritional properties are enhanced, thanks to the fact that microorganisms synthesize vitamins, minerals, and bioactive peptides [22, 23], among others, which are beneficial to human health. This is why the evaluation of fermentation processes such as milk fermentation with *Aspergillus niger* arouses interest.

On the other hand, worldwide interest has increased in topics related to conditions caused by oxidative stress since this is related to the development and onset of various human diseases [24, 25], including atherosclerosis [26], Alzheimer's disease [27] and cancer [28], among others.

To analyze the previous problem, a solution was found by evaluating the antioxidant activity through the DPPH▪ , ABTS▪<sup>+</sup> methodologies, evaluation of thiol groups, and evaluation of superoxide dismutase activity; all these analyses were performed by spectrophotometric techniques in 96-well plates, in the presence of peptide extracts obtained during the fermentation of lactic serum with *Aspergillus niger*.

#### **2. Methodology**

#### **2.1 Obtaining the inoculum**

The inoculum of *A. niger* CMPUJH002, provided by the collection of microorganisms of the Universidad Pontifica Javeriana, was obtained by spiking on potato dextrose agar (PDA) at 37°C for 7 days. After its use, it was preserved by the method of spore suspension in glycerol of the laboratory of the biotechnology research group natural products of the Universidad Tecnologica de Pereira (GB-PN, UTP).

#### **2.2 Lactic fermentation**

To obtain the peptide extracts, the methodology proposed by Channe & Shewale [29] was used, where lactic serum (enriched and prepared medium) was prepared and sterilized (**Table 1**), as a substrate for *A. niger*, in a 1 L Erlenmeyer, covered with vinipelt plastic. Fermentation was carried out over a period of 8 days, with 400 mL of substrate and 1.6 cm2 of inoculum of *A. niger* grown on PDA agar after 7 days of growth at 37°C. The fermentation process was carried out with the following conditions pH 4.5, temperature 30°C, and an agitation of 71.76 1.25 rpm, leaving a headspace of 60%. This assay was assembled in quadruplicate, coding each extract

*Whey Protein Fermentation with* Aspergillus niger*: Source of Antioxidant Peptides DOI: http://dx.doi.org/10.5772/intechopen.111895*


#### **Table 1.**

*Composition of lactic whey fermentation medium enriched with* Aspergillus niger *taken from Channe & Shewale [29].*

with an F indicating fermentation and a letter D followed by a number indicating the day it was collected.

#### **2.3 Partial purification of peptide extracts**

The extracts collected daily were added 10% trichloroacetic acid [30], in equal proportion with respect to the sample (1, 1), in order to precipitate the large proteins and eliminate contaminants, then the precipitation of low molecular weight proteins was carried out by adding 50 mM ZnSO4/Acetone 80% to the sample in a ratio of 0.25:0.25:0.5 with respect to the sample, then it was placed in a refrigerator at 2°C for 24 hours for subsequent centrifugation at 5000 rpm at 3°C [31].

#### **2.4 Antioxidant activity against DPPH radicals**

A solution of DPPH▪ 20 mg/L in methanol was prepared, and 100 μL of this solution was added to each of the wells containing 25 μL of the extract to be evaluated; it was left to react for 30 minutes in darkness and after this time the absorbance was read at 517 nm [25] using the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer. This analysis was carried out in quadruplicate.

#### **2.5 ABTS <sup>+</sup> antioxidant activity**

A solution of ABTS 3.5 mM and potassium persulfate 1.25 mM dissolved in H2O was prepared (this mixture was made at least 12 hours before its use), after this time the absorbance of the ABTS▪ + solution was adjusted to 0.7+/�0.02 units at 732 nm with ethanol; 194 μL of this solution was transferred to the well containing 6 μL of the extract to be evaluated, and it was left to react for 30 minutes in darkness to read its absorbance at 732 nm, in the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer. This analysis was carried out in quadruplicate [32].

From the absorbances obtained, the percentage of antioxidant activity is determined with the following equation described by Perez and coworkers [25, 32].

$$\text{\%Antioxidant Activity} = \left[ \frac{A\_{control\ (-)} - (A\_{extract} - A\_{white\ extract)}}{A\_{control}} \right] \text{\because 100} \tag{1}$$

Where,

Aextract: Absorbance of the extracts. Ablanco extracto: Absorbance of the blank of the extracts. Acontrol (�): Absorbance of the negative control.

#### **2.6 Content analysis of thiol groups (-SH)**

For the quantification of thiols, the reaction of the samples with Ellmann's reagent (DNTB (5,5'-Dithiobis(2-nitrobenzoic acid)) was carried out, taking 95 μL of sample with 30 μL of Na2HPO4 buffer, 0.5 M pH 7.0, with 125 μL DNTB at 10 nM incubating for 15 minutes and reading their absorbances at 412 nm, on Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer equipment; the calibration curve was prepared at different concentrations using glutathione enzyme, Na2HPO4 buffer and sulfoanilic acid 20%(m/v), 125 μL of each standard was taken and analyzing as samples [33].

#### **2.7 Quantification of the enzyme superoxide dismutase (SOD)**

The method used for the determination of superoxide dismutase was based on the protocol carried out by Betancur and Mosquera [33], where a hydroxylamine calibration curve is constructed that interacts with a xanthine/xanthine oxidase system as a source of generation of a superoxide anion flux that oxidizes hydroxylamine to nitrite for a subsequent measurement of nitrite concentration by UV/visible spectrometry.

To perform the analysis, sample preparation was done with the addition of 150 μL KH2PO4 buffer, pH 7.8, 40 μL of deionized water, 15 μL of xanthine, 15 μL hydroxylamine hydrochloride 1 mM, and 75 μL xanthine oxidase (0.2 mg protein/mL); the standards for the curve were prepared with phosphate buffer, water, xanthine, xanthine oxidase, hydroxylamine chloride, and the enzyme (SOD), and the system was left to react for 20 minutes in the dark, Then the reaction was stopped in an ice bath to add 100 μL of 19 mM sulfanilic acid and 7 mM α-naphthylamine, after which 100 μL of sample or standard was added, incubated for 20 minutes and its absorbance was measured at 529 nm in the Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer.

#### **2.8 Protein profile of peptide extracts by denaturing electrophoresis**

Electrophoresis was carried out according to Bio-Rad guidelines under denaturing conditions. Separation gels were 15% SDS-polyacrylamide (30% bis-acrylamide, 1.5 M Tris-HCl pH 8.8, 10% ammonium persulfate, 0.04% tetramethylethylenediamine (TEMED), 10% SDS, and distilled water to final volume) and 5% SDS-polyacrylamide stacking gels (30% bis-acrylamide, 1 M Tris-HCl pH 6.8, 10% ammonium persulfate, 0.1% TEMED, 10% sodium dodecyl sulfate (SDS), and distilled water. The solutions were cast in mini protean (Bio-Rad) with the addition of ammonium persulfate and TEMED to polymerize the gels (1 mm thick).

*Whey Protein Fermentation with* Aspergillus niger*: Source of Antioxidant Peptides DOI: http://dx.doi.org/10.5772/intechopen.111895*

The gels were loaded with 5% concentration samples, which were diluted in 1:1 reducing buffer (Tris HCl (63 mM), glycerol (10%), SDS (2%), bromophenol blue (0.0025%) pH 6.8), heated at 85°C for 2 to 5 minutes; the electrophoresis was performed at room temperature (24 and 27°C) using a constant current of 35 mA at 120 V for approximately 5 h. Once the run was completed, the gels were washed two times with deionized water and stained with a 0.1% Coomassie brilliant blue R-250 solution with 40% methanol and 10% acetic acid for 12 h with gentle agitation and then destained 2 times for 20 minutes with a 25% ethanol and 8% acetic acid solution [34].

#### **2.9 Statistical analysis**

For the statistical treatment of the data obtained for antioxidant and antibacterial activity, a one-way ANOVA analysis by replicates with a confidence level of 95% was carried out. All statistical analysis was carried out using GraphPad Prism 8.4.3 software.

#### **3. Results and discussion**

#### **3.1 Antioxidant evaluation by means of the DPPH-radical**

The DPPH- decolorization methodology carried out allowed establishing that the extracts evaluated have an antioxidant capacity that inhibits the DPPH radical by more than 50%; however, these values are lower than the percentage of antioxidant capacity of the hydroquinone positive control (**Figure 1**). Likewise, these results are lower, compared to other similar works, where the antioxidant activity on DPPHpresented by peptides derived from proteases of *Aspergillus oryzae* and *Aspergillus flavipes* species in milk, exceeded 90% [9], through the same method, since the method of obtaining the peptides was carried out from the already purified fungal proteases, which leads to their obtaining in a targeted manner, without competition from other enzymes and without the generation of secondary reactions due to the use of the complete metabolism of the microorganism [35].

The results are attributed to the activity coming from the phenolic compounds, which are mainly responsible for the antioxidant activity, being one of the reasons why it is not a recommended technique for samples of biological origin [36], which is why the evaluation of ABTS + cation radical decolorization was carried out as a complementary technique to contrast the results.

#### **3.2 ABTS <sup>+</sup> antioxidant evaluation**

In this method, the evaluation of antioxidant activity exceeds 90% in almost all cases and can be considered a good antioxidant because the activity significantly exceeds the positive control with equal concentration (1000 ppm) (**Figure 2**).

Although oxidative stress is generated when the oxide-reduction homeostasis state of the cell becomes unbalanced because the antioxidant and prooxidant counterparts are altered. Aerobic organisms activate their defense mechanisms such as the secretion and action of glutathione, which is a tripeptide, composed of glutamate, cysteine, and glycine, that is used at the biological level for the regulation of this oxidation, which is why it is

**Figure 1.**

*One-way ANOVA of the determination of antioxidant activity (%AA) by DPPH of fermentations (F) 1, 2, 3, and 4 with their respective days (D) 1, 2, 3, 4, 5, 6, and 7. A) Fermentation 1, b) fermentation 2, c) fermentation 3, and d) fermentation 4. Equal symbol (a; b; c) indicates that there are no significant differences, and equal sign indicates that there are significant differences (P < 0.05).*

considered the universal antioxidant, in fact, it has been attributed that the proper functioning of most other antioxidants is due to the presence of glutathione [37].

Therefore, in addition to the biological activities proposed, the determination of thiols (-SH) in the different peptide extracts was also carried out through the construction of a calibration curve as shown in **Figure 3**.

This analysis provided the plot eq. Y = 0.03769X + 0.02248, with an r2 of 0.9927 and a p < 0.05, to determine the concentration of glutathione conferring antioxidant activity to the peptides present in each sample, as synthesized in **Table 2**.

To complement the results obtained on the antioxidant potential of peptide extracts, the evaluation of superoxide dismutases (SOD), whose main function is the defense of aerobic organisms such as yeasts and filamentous fungi, such as *Aspergillus oryzae*, was carried out, where through transcriptomic analysis the expression of enzymes, such as catalase, glutathione peroxidase, and superoxide dismutase, has been demonstrated to regulate the concentration of oxidizing agents, such as oxygen free radicals, especially superoxide anion radicals [9]. The results obtained from the

**Figure 2.**

*One-way ANOVA ABTS <sup>+</sup> of fermentations (F) 1, 2, 3, and 4 with their respective days (D) 1, 2, 3, 4, 5, 6, and 7. A) Fermentation 1, b) fermentation 2, c) fermentation 3, and d) fermentation 4. Equal symbol (a; b; c) indicates no difference means, and equal sign indicates significant differences (p < 0.05).*

**Figure 3.** *Calibration curve for thiol (-SH) quantification.*


#### **Table 2.**

*SH concentration (mmol/μL) of the different peptide extracts.*

radical uptake assessment using the superoxide dismutase model are given in **Figure 4**.

It has been identified that the antioxidant capacity of milk and its derivatives are mainly due to sulfur-rich amino acids such as tyrosine and cysteine, vitamins A and E, carotenoids, and enzyme systems such as the enzyme superoxide dismutase (SOD), which are useful so that superoxide radicals (O2–), hydroxyl radicals, and peroxide radicals can be inhibited [9].

This analysis provided the equation of the graph Y = 0.3146X - 0.2602 with an r<sup>2</sup> equal to 0.9933 and a p < 0.05; to determine the concentration of SOD units present in each sample, as synthesized in **Table 3**, which has the corresponding calculations of the transformation from -log (SOD units) to SOD units.

This analysis revealed the presence of peptides with SOD-type antioxidant character; although a difference in this concentration is evident in the different fermentations, the reason for these differences cannot be discerned without other analyses,

**Figure 4.** *Calibration curve for SOD determination, log (SOD units) vs. absorbance.*

*Whey Protein Fermentation with* Aspergillus niger*: Source of Antioxidant Peptides DOI: http://dx.doi.org/10.5772/intechopen.111895*


#### **Table 3.**

*Concentrations in SOD units of the different peptide extracts.*

which is why the analysis support is required as a characterization of the peptide extracts.

However, taking into account reports by other authors on the antioxidant activity of raw bovine milk with respect to SOD concentration of 0.92 to 3 units/mL and low concentrations of -SH [38, 39], a decrease is evidenced by the elimination of proteins of higher molecular weight removed during partial purification, that is, concentrated proteins of low molecular weight are good antioxidant agents.

#### **3.3 Analysis of the peptide profile of the extracts by SDS-page electrophoresis**

**Figure 5** shows the peptide profile exhibited by the peptides obtained, where several fragments of molecular weight above 10 KDa are evident, which led to think that there are still precursor proteins within the extracts; these proteins have already been reported in other studies [40] with their molecular weights as expressed in **Table 4**; therefore, the purification process was not effective in eliminating these high molecular weight proteins.

Likewise, although the technique used does not allow the characterization of peptides of molecular weights lower than 10 KDa, it was the only one that allowed the

**Figure 5.** *Electrophoresis,Tris-glycine-SDS for polyacrylamide gel electrophoresis.*


#### **Table 4.**

*Molecular weights in KDa of some important proteins in bovine milk.*

visualization of bands of protein origin because techniques, such as SDS-triscin [7], did not allow the visualization of bands, as well as silver staining, the result of the electrophoresis can be seen in **Figure 5**; although theoretically it is known that the protein concentration in a sample should be greater than 0.5 mg/mL [34], the samples analyzed have a variable composition and of different concentrations.

#### **4. Conclusions**

Antioxidant bioactive peptides have become a very valuable tool in both the food and pharmacological industries due to their ability to act as antioxidants, and thus combat oxidative stress in the human body. These peptides can be obtained from proteins by fermentation processes with microorganisms and enzymes.

In this case, *Aspergillus niger* was used as an enzymatic source to obtain antioxidant peptides from lactic serum. The results obtained indicate that the extracts obtained from this fermentation process have a promising antioxidant activity, which makes them a very interesting nutraceutical substrate.

Importantly, the antioxidant activity of these extracts can be attributed to the presence of thiol groups and positive SOD activity. The ability to catalyze the dismutation of superoxide anion into hydrogen peroxide and molecular oxygen is a very important characteristic of these extracts as it confers them a greater capacity to combat oxidative stress in the human body.

Therefore, the results obtained indicate that the use of *Aspergillus niger* as an enzymatic source for obtaining antioxidant peptides from lactic serum is a very valuable tool in the food and pharmacological industry. These extracts have promising antioxidant activity and are a very interesting source of nutrients to combat oxidative stress in the human body.

#### **Acknowledgements**

To the Vice-Rectory of Research of the Universidad Tecnológica de Pereira, for financing project E9-20-3 and to the Universidad de Manizales, where the characterization of the extracts by electrophoresis was carried out.

*Whey Protein Fermentation with* Aspergillus niger*: Source of Antioxidant Peptides DOI: http://dx.doi.org/10.5772/intechopen.111895*

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marcela Patricia Gomez Rojas and Oscar Marino Mosquera Martinez\* Universidad Tecnológica de Pereira, Pereira, Colombia

\*Address all correspondence to: omosquer@utp.edu.co

© 2023 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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### *Edited by Mehdi Razzaghi-Abyaneh, Mahendra Rai and Masoomeh Shams-Ghahfarokhi*

This book is divided into five sections and ten chapters, highlighting recent advances in *Aspergillus* and aspergillosis from pathogenicity to novel diagnosis based on biosensors and metagenomic next-generation sequencing, mechanisms of antifungal drug resistance, *Aspergillus–*human interactions, immunopathogenesis of invasive aspergillosis, post-viral aspergillosis, treatment strategies, and the importance of beneficial and harmful metabolites of *Aspergillus* in public health and industry. This book presents cutting-edge research on *Aspergillus* along with useful information for mycologists, microbiologists, toxicologists, plant pathologists, and pharmacologists who may be interested in understanding the impact, significance, and recent advances within the genus *Aspergillus* that have not been critically noticed elsewhere.

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*Aspergillus*

and Aspergillosis - Advances in Genomics, Drug Development,

Diagnosis and Treatment

IntechOpen Series

Infectious Diseases, Volume 25

*Aspergillus* and Aspergillosis

Advances in Genomics, Drug Development,

Diagnosis and Treatment

*Edited by Mehdi Razzaghi-Abyaneh, Mahendra Rai* 

*and Masoomeh Shams-Ghahfarokhi*