Recent technological advances in mechanism, toxicity, and food perspectives of enzyme-mediated aflatoxin degradation

Abstract

Aflatoxins are carcinogenic secondary metabolites produced by Aspergillus section Flavi that contaminates a wide variety of food and feed products and is responsible for serious health and economic consequences. Fermented foods are prepared with a wide variety of substrates over a long fermentation time and are thus vulnerable to contamination by aflatoxin-producing fungi, leading to the production of aflatoxin B1. The mitigation and control of aflatoxin is currently a prime focus for developing safe aflatoxin-free food. This review summarizes the role of major aflatoxin-degrading enzymes such as laccase, peroxidase, and lactonase, and microorganisms in the context of their application in food. A putative mechanism of enzyme-mediated aflatoxin degradation and toxicity evaluation of the degraded products are also extensively discussed to evaluate the safety of degradation processes for food applications. The review also describes aflatoxin-degrading microorganisms isolated from fermented products and investigates their applicability in food as aflatoxin preventing agents. Furthermore, a summary of recent technological advancements in protein engineering, nanozymes, in silico and statistical optimization approaches are explored to improve the industrial applicability of aflatoxin-degrading enzymes.

Keywords:

• Aflatoxins
• degradation
• enzyme
• microorganisms
• fermented food
• toxicity

Introduction

Mycotoxins contaminate various food ingredients and products, posing a serious health risk to humans and animals. A number of mycotoxins, such as aflatoxins, ochratoxin A, zearalenone, fumonisins, patulin, and deoxynivalenol, are reported toxic to humans, and thus threaten food safety (Stroka and Gonçalves 2019). Among them, aflatoxins are produced by various Aspergillus species, such as Aspergillus flavus and Aspergillus parasiticus (Khaneghah et al. 2018). Aspergillus flavus mainly produces aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), whereas Aspergillus parasiticus produces aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). AFB1 is classified as a group I carcinogen according to the International Agency for Research on Cancer (Ostry et al. 2017). Aflatoxins are quite stable under most conditions, thus aggravating health risks and financial losses.

Different types of food materials, such as spices, nuts, cereals, oil seeds, milk, and legumes, can be contaminated with aflatoxins (Liu, Galani Yamdeu et al. 2020). These food materials are an integral part of food products; therefore, aflatoxin in raw food materials ultimately contaminates the final food product, leading to human exposure. The Food and Agriculture Organization of the United Nations stated that approximately 25% of food materials worldwide are affected by aflatoxin contamination (Eskola et al. 2020). Therefore, aflatoxin contamination is a serious problem in food supplies worldwide.

Three major strategies, such as physical, chemical, and biological methods, are typically used for aflatoxin detoxification in food and feed products (Peng et al. 2018). Physical methods include sorting, milling, cleaning, microwave heating, cooking, roasting, ultraviolet irradiation, gamma irradiation, and photocatalysis (Pankaj, Shi, and Keener 2018), whereas chemical methods involve aflatoxin conversion through chemical reactions, such as alkaline hydrolysis, ammoniation, ozonation, and peroxidation (Pankaj, Shi, and Keener 2018). Physical and chemical methods have been found to be effective at detoxifying mycotoxins; however, the toxicity of end products and food characteristics, such as nutritive value, organoleptic properties, and palatability, remain questionable. Therefore, biological detoxification methods have become the most promising alternative for aflatoxin decontamination in feed and food products.

Biological detoxification through microorganisms can be conducted under mild conditions that minimize the loss of nutritive value of food together with other food characteristics (Alberts et al. 2009; Samuel, Sivaramakrishna, and Mehta 2014). Several mechanisms, such as simple binding and/or adsorption by microbial cell surfaces and enzymatic degradation, are involved in the biological detoxification of aflatoxins. Simple adsorption and binding do not necessarily destroy the structure of aflatoxin but reduce gastrointestinal aflatoxin absorption by modifying their structure, thereby reducing health hazards. However, adsorption and binding are reversible processes that may release aflatoxins under certain conditions, and the reversibility of the process depends on the adsorbent properties (Solis-Cruz et al. 2018). In contrast, enzymatic biodegradation modifies aflatoxin structures into less or nontoxic structures.

The present review mainly focuses on enzyme-mediated aflatoxin degradation and explores several related aspects, including aflatoxin-degrading enzymes and microorganisms, mechanisms of aflatoxin degradation by microbial enzymes, and toxicity of degraded products. To the best of our knowledge, a review of the literature that focuses on enzyme-mediated aflatoxin degradation by microorganisms is lacking. Biochemical pathways for aflatoxin degradation are proposed based on the structures of the metabolites produced by enzymatic degradation. Recent technological advances in nanotechnology, protein engineering, and bioinformatics that improve the catalytic efficiency, stability, and activity of aflatoxin-degrading enzymes, making them suitable candidates for food applications, are also reviewed. In addition, food applications are discussed, such as controlling aflatoxin levels in fermented food using microorganisms and their products and aflatoxin-degrading microorganisms isolated from food products. The review also insight the feasibility and importance of enzymatic aflatoxin degradation in fermented foods. The prime significance of using enzymes in fermented foods is due to the high specificity of enzyme-catalyzed reactions that selectively act on the aflatoxin without interacting with food components and thus preserve the essence of food. Finally, the techno-economic feasibility of the industrial application of enzymes to mitigate aflatoxin contamination is also discussed.

Microorganisms involved in aflatoxin degradation

Microbial aflatoxin degradation was first observed in Nocardia corynebacterioides in the late 1960s. Since then, several microorganisms that can detoxify aflatoxins have been identified, such as bacteria, Actinobacteria, Lactobacillus, yeast, fungi, and edible mushrooms. Lactobacillus and yeast detoxify aflatoxins in food products mainly through binding and adsorption (Luo et al. 2020). Several bacteria belonging to the Bacillus, Actinobacteria, and α- and β-proteobacteria groups have been shown their efficiency to degrade aflatoxins. Several Bacillus species, including Bacillus subtilis (Afsharmanesh et al. 2018; Suresh et al. 2020), Bacillus licheniformis (Rao et al. 2017), Bacillus velenzensis (Shu et al. 2018), Bacillus cereus, and Bacillus mojavensis (Pereyra, Martínez, and Cavaglieri 2019), have been found to degrade aflatoxin. Moreover, other bacteria, such as Pseudomonas fluorescens, Pseudomonas anguilliseptica, and Staphylococcus sp. have been identified as AFB1 degraders (Adebo et al. 2016). Among actinomycetes, Streptomyces is the genus that has been studied the most in relation to aflatoxin degradation. Eshelli et al. (2015) identified three actinomycetes species, Streptomyces lividans, Streptomyces aureofaciens, and Rhodococcus erythropolis, and Harkai et al. (2016) identified Streptomyces cacaoi, that could degrade AFB1. Furthermore, fungi are known for their large repertoire of extracellular enzymes and are important aflatoxin degraders. Fungal isolates of Phanerochaete sordida (Wang et al. 2011), Aspergillus niger (Zhang et al. 2014), Irpex lacteus, Phanerochaete chrysosporium, Ceriporiopsis subvermispora (Wang et al. 2019), Bjerkandera adusta (Choo et al. 2021), Trametes versicolor (Suresh et al. 2020), and Fusarium sp. WCQ3361 (Wang, Li et al. 2017) have been reported to degrade AFB1. Noteworthily, even though Aspergillus flavus is known for aflatoxin production, some atoxigenic Aspergillus flavus strains have been identified as aflatoxin degraders. For example, Xing et al. (2017) isolated atoxigenic Aspergillus flavus JZ2 and Aspergillus flavus GZ15 from peanut fields and observed effective AFB1 degradation. Furthermore, some edible mushrooms such as Armillariella tabescens (Xu, Xie et al. 2017), Pleurotus ostreatus (Jackson and Pryor 2017), and Pleurotus pulmonarius (Loi et al. 2016) have also been identified to have an aflatoxin degradation ability. Recently, Söylemez, Yamaç, and Yıldız (2020) reported that the mushroom Panus neostrigosus could efficiently degrade aflatoxin.

Aflatoxin-degrading enzymes

A number of aflatoxin-degrading enzymes have been extracted and purified from various microorganisms, including bacteria, Actinobacteria, and filamentous fungi (Loi et al. 2018; Sangare et al. 2014; Wu et al. 2009). The major enzyme groups of laccases, peroxidases, reductases, and lactonases have been documented for aflatoxin degradation.

Laccases

Laccases belong to a class of multicopper oxidases and catalyze the oxidation of several polycyclic aromatic hydrocarbons, phenolic compounds, and aromatic amines to other less or nontoxic compounds with the simultaneous reduction of molecular oxygen to water (Loi et al. 2016). Alberts et al. (2009) reported AFB1 degradation by the culture supernatant of Trametes versicolor, Bjerkandera adusta, Peniophora sp., Phanerochaete chrysosporium, and Pleurotus ostreatus with laccase activity and the loss of mutagenicity and fluorescence of AFB1. Alberts et al. (2009) used pure laccase from Trametes versicolor for AFB1 degradation and observed 87.34% degradation after 72 h of incubation using 1 U/mL of enzyme, whereas Zeinvand-Lorestani et al. (2015) used purified laccase from Trametes versicolor and observed 67% degradation after 48 h of incubation using 30 U/mL of enzyme. Loi et al. (2016, 2018) observed that Pleurotus pulmonarius Lac2 and Pleurotus eryngii Ery4 could degrade AFB1 up to 90% in the presence of redox mediators. Recently, Song et al. (2021) identified Laccase2 from Pleurotus pulmonarius and showed 99.82% AFB1 degradation at 37 °C and pH 7.0 after 1 h of incubation in the presence of the redox mediator acetosyringone. Laccase from Trametes versicolor is capable of catalyzing the direct oxidation of AFB1 (Alberts et al. 2009; Zeinvand-Lorestani et al. 2015); however, laccase from Pleurotus pulmonarius Lac2 and Pleurotus eryngii Ery4 requires redox mediators to achieve AFB1 oxidation or aflatoxin degradation (Loi et al. 2016, 2018). Bacterial laccases exhibit characteristics superior to fungal laccases, such as high thermostability, broad substrate spectrum, wide pH range, and tolerance to alkaline conditions, which make them more suitable candidates for aflatoxin degradation (Guan et al. 2018). Recently, Guo et al. (2020) identified a highly thermostable bacterial laccase from Bacillus licheniformis with a half-life of 1 h at 70 °C and the ability to degrade 70% AFB1 within 30 min.

Peroxidases

Laccases require additional redox mediators for efficient mycotoxin degradation because of their low redox potential (≤0.8 V) (Loi et al. 2018), whereas peroxidases do not require additional redox mediators because they carry a high redox potential (≥1.0 V) (Sáez-Jiménez et al. 2015). Peroxidases have been identified as mycotoxin biodegraders, (including AFB1) by various researchers (Loi et al. 2020; Wang et al. 2011; Yehia 2014). Wang et al. (2011) observed 86% AFB1 degradation with purified manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624, whereas Yehia (2014) observed 67% AFB1 degradation from Pleurotus ostreatus. Wang et al. (2019) reported the AFB1 degradation by manganese peroxidase that occurs through free radical attack, which is similar to the mechanism for dye decolorization. Two manganese peroxidases from Phlebia sp. Nfb19 and Bjerkandera adusta (Tonin et al. 2017) and recombinant N246A variant of dye88 decolorizing DypB (Rh_DypB) from Rhodococcus jostii (Vignali et al. 2018) were compared for their AFB1 degradation capability by Loi et al. (2020); as results, Rh_DypB was found to have the highest AFB1 degradation efficiency (96%). Moreover, Marimón Sibaja et al. (2019) observed 65% aflatoxin M1 (AFM1) degradation and 97% AFB1 degradation in milk with horse radish peroxidase at 30 °C after 8 h of incubation.

F₄₂₀-dependent reductases

The F_420-dependent reductases reduce α,β-unsaturated esters, leading to the destabilization and ultimate cleavage of the lactone ring. Taylor et al. (2010) identified that Mycobacterium smegmatis produces F₄₂₀/H₂-dependent reductases that catalyze aflatoxin degradation. Two categories, F₄₂₀-dependent reductases-A and F₄₂₀-dependent reductases-B, were recognized for aflatoxin degradation; the former were 100 times more active than the latter. As a low redox potential cofactor, F₄₂₀ is limited to Actinomycetales, certain Proteobacteria, and Archaea. Therefore, it is assumed that these F₄₂₀-dependent reductases may be responsible for the degradation capability of Actinomycetales (Lapalikar et al. 2012).

Lactonases

Lactonases are a family of enzymes known to degrade AFB1 by the destruction of the lactone ring of aflatoxin. Thus, opening the lactone ring is effective at reducing aflatoxin toxicity. Bacillus cereus, Bacillus mycoides, Bacillus anthracis, Bacillus thurigensis, Bacillus amyloliquefaciens, Bacillus weihenstephanensis, and Bacillus subtilis are known to produce N-acyl homoserine lactone (AHL) lactonase, which cleaves the lactone ring by targeting its ester bond (Pereyra, Martínez, and Cavaglieri 2019). Pereyra, Martínez, and Cavaglieri (2019) reported that certain Bacillus species possessed the aii gene, encoding the AHL lactonase responsible for AFB1 degradation.

Other unidentified enzymes

In addition to the enzymes discussed above, other enzymes have been reported to degrade aflatoxin. Zhao et al. (2011) reported the myxobacteria aflatoxin degradation enzyme produced by Myxococcus fulvus ANSM068, with a degradation capability of 96.96% for AFG1 and 95.80% for AFM1. A thermostable Bacillus aflatoxin-degrading enzyme was purified from Bacillus shackletonii that reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively (Xu, Eisa Ahmed et al. 2017). The Bacillus aflatoxin-degrading enzyme was an oxidoreductase and its degradation activity was significantly improved by Cu²⁺ ions. Additionally, Xu, Xie et al. (2017) reported aflatoxin oxidase, which is mainly produced by the medicinal and edible fungi Armillariella tabescens. Previously, aflatoxin oxidase was known as aflatoxin-detoxizyme. Subsequently, aflatoxin-detoxizyme was identified as an oxidase enzyme and renamed aflatoxin oxidase. The protein structure of aflatoxin oxidase indicates that it is a new oxidase that differs from other reported aflatoxin oxidases, such as laccase and horseradish peroxidase. Recently, Pantoea aflatoxin degradation enzyme purified from Pantoea sp. T6 showed AFB1 degradation activity in cell-free supernatant (68.30%) (Xie, Wang, and Zhang 2019).

Mechanism of enzymatic aflatoxin degradation

The mechanism of the enzyme degradation pathway for aflatoxin has not been sufficiently studied. Research has shown the formation of various metabolites, although most studies failed to establish clear sequential changes during degradation.

Laccases are the most studied enzymes for aflatoxin detoxification (Alberts et al. 2009; Zhang et al. 2014). Laccases act on AFB1 in two ways. Firstly, laccase can act on the terminal furan ring of AFB1, leading to the formation of AFB1-epoxide, which is further converted to AFB1 dihydrodiol, rendering AFB1 to a less toxic state. Secondly, laccase may directly open the lactone ring by introducing hydroxyl groups at the carbon 10 and 11 positions in AFB1 (Figure 1). Wang et al. (2011) revealed that the laccase/manganese peroxidase-mediated oxidation of double bond in the furan ring of AFB1 leads to AFB1-8,9-epoxide formation, which further undergoes hydrolysis to form AFB1-8,9-dihydrodiol (Figure 1a). Similarly, Zaccaria et al. (2020) studied AFB1 conversion by laccase using quantum mechanism and other approaches and observed that in the presence of an environmental hydrogen atom, AFB1 undergoes structural rearrangement to form epoxide in the terminal ring, which is further converted into dihydrodiol. Recently, Guo et al. (2020) demonstrated the role of Cot A laccase from Bacillus licheniformis to oxidize AFB1. Cot A catalyzes the C3-hydroxylation of AFB1 by converting it into two isomeric forms, aflatoxin Q1 (AFQ1) and epi-AFQ1 (Figure 1b). A similar conversion of AFB1 to AFQ1 was documented by Loi et al. (2020) using Rh_Dyp peroxidase.

Figure 1. Laccase-mediated aflatoxin B1 degradation pathway. (a) Attack on the lactone and furan ring; (b) C3-hydroxylation in aflatoxin B1 to produce two isomeric compounds aflatoxin Q1 and epi-aflatoxin Q1.

Borgomano (2015) demonstrated the degradation of AFB1 into different metabolites in response to partially purified laccase from Coriolus hirsutus. The mass/charge ratio (m/z = 327.254) of the major product C₁₇H₁₁O₇ indicated the presence of an additional oxygen atom compared to that in AFB1, signifying the epoxidation of the terminal furan ring double bond (Figure 2). Another compound, C₁₆H₁₂O₅ (m/z = 285.076), identified as 2,3,3a,6a-tetrahydrofuro [2,3-b]furan 8-methoxy-1,2-dihydro-3H-cyclopenta[b]benzofuran-3-one, had one carbon and oxygen atom less than AFB1 (Figure 2). The removal of carbon and oxygen from AFB1 most probably occurs at the lactone ring. The metabolite C₁₅H₁₁O₄ (m/z 255.185) (Figure 2) was identified as 1,2,5a,8a-tetrahydro-10-H-cyclobuta[c]furo[3′,2′:4,5]furo[2,3-h]chromen-10-one, which may be derived from AFB1-dihydrodiol, where dehydration of the diol group may provoke methoxy group alteration, followed by the removal of two carbon and oxygen atoms. Alternatively C₁₅H₁₁O₄ (m/z = 255.185) can be formed by another tentative pathway that could proceed like the hydroxylation of AFB1 to form aflatoxin B2a (AFB2a), followed by the loss of one H₂O molecule, which supports the removal of methoxy and CO groups (Figure 2). Similarly, another tentative pathway may be the conversion of AFB1 to AFB2a, followed by the removal of two CO and H₂O molecules, leading to the formation of C₁₅H₁₁O₄ (m/z 255.185; 2,3,3a,6a-tetrahydrofuro[2,3-b]furan-1,2-dihydrocyclobuta[b]benzofuran-7-carbaldehyde) (Figure 2). In addition, two metabolites, 2,3,3a,4,5,8a-hexahydrofuro[2,3-b]benzofuran-2,6-diol (C₁₀H₁₃O₄, m/z = 196.86) and 3a,4,5,8a-tetrahydrofuro[2,3-b]benzofuran-2,6-diol (C₁₀H₁₁O₄, m/z = 195.194), were identified that were believed to be produced from aflatoxin P1 (AFP1) by O-demethylation and AFB1 hydroxylation (Figure 2). Borgomano (2015) concluded that dehydrogenation, hydroxylation, dehydration, keto group reduction, and the removal of carbon, oxygen, and methyl motifs are the principal mechanisms induced by laccase.

Figure 2. Degradation products of aflatoxin B1 by partially purified laccase from Coriolus hirsutus (Borgomano 2015).

The cell-free extract of Trametes versicolor exhibited AFB1 degradation ability, wherein the degradation products appeared as cleaved lactone or furan rings of AFB1, mainly because of the involvement of laccase (Suresh et al. 2020). Metabolites such as C₁₆H₁₈O₅, C₆H₁₂O, and C₉H₁₈O₂ were detected with lack of lactone ring, whereas the metabolites of C₁₂H₈O₄, C₆H₁₂O, C₉H₁₄O₄, and C₉H₁₈O₂ showed complete cleavage of the furan ring (Suresh et al. 2020). In contrast, the metabolites of C₁₇H₁₄O₇, C₁₆H₁₈O₅, and C₁₆H₁₂O₇ were observed with modified furan by redox reactions (Table 1) (Suresh et al. 2020). Consistent with the results obtained from Trametes versicolor, the cell-free extract of Bacillus subtilis also produced different degraded metabolites, such as C₁₇H₂₀O₇, C₉H₁₀O₃, C₉H₁₄O₂, C₈H₁₂O, C₁₆H₁₂O₂, and C₆H₁₀, from AFB1 through a series of redox reactions (i.e., hydroxylation, hydrogenation, and dehydrogenation) (Suresh et al. 2020).

Table 1. A brief account of enzyme-mediated aflatoxin degradation products and their toxicity.

Expected aflatoxin-degrading enzymes	Expected structure of degrdation		Target moiety of aflatoxin B1	Toxicity of degraded products		Microorganism involved in aflatoxin degradation	References
Lactonase, reductase	(1) C15H20O6	(2) C11H16O4	Lactone ring and difuran ring	Removal of the lactone ring reduces its mutagenicity by 450‐fold and acute toxicity by 18‐fold.		Aspergillus niger, atoxigenicAspergillus flavus	Xing et al. 2017
Reductase	C17H15O6		Lactone ring	Unstable lactone ring leads toward loss of toxicity.	Aspergillus niger, atoxigenicAspergillus flavus		Taylor et al. 2010
Laccase, oxidoreductase	(1) C17H14O7	(2) C16H18O5	Furofuran double bond and lactone ring	Product 2 lacks the lactone ring, while products 1, 2, 4, 5, and 6 are either completely degraded or appeared to have a partially modified furan ring. These products show reduced toxicity.	Termetes versicolor		Suresh et al. 2020
	(3) C16H10O6	(4) C12H8O4
	(5) C16H12O7	(6) C14H10O4
Hydrolases, dehydrogenases, bacilysin, oxidoreductase N-acyl homoserine lactonase	(1) C17H12O8 (3) C16H12O5	(2) C17H20O7	Terminal furan and lactone ring	Product 2 lacks lactone ring and products 1 and 2 are either completely degraded or partially modified furan ring, leading to loss of toxicity.	Bacillus sp.		Suresh et al. 2020
Unidentified enzymes present in cell-free extract of Tetragenococcus halophilus	(1) C14H10O4	(2) C18H16O8	Lactone ring and difuran ring	Products are expected to reduce toxicity due to removal of terminal double bond in furan ring and lactone ring.	Tetragenococcus halophilus		Li et al. 2018a
	(3) C14H12O3	(4) C16H20O4
	(5) C14H16O2	(6) C14H20O2
Unidentified multiple enzymes in cell-free extract	(1)Aflatoxin D1 (C16H14O5)	(2) Aflatoxin D2 (C11H10O4)	Terminal furan ring and lactone ring	Toxicity is very less toward HeLa cells due to loss of lactone ring and furan ring.	Pseudomonas putida, Bacillus subtilis, Rhodococcus erythropolis		Afsharmanesh et al. 2018; Eshelli et al. 2015; Samuel, Sivaramakrishna, and Mehta 2014
	(3) Aflatoxin D3 (C8H4O3)
Unidentified intracellular as well as extracellular enzymes	(1) C14H10O4	(2) C14H12O3	Lactone ring	Products 1, 2, and 3 have lower toxic toward brine shrimps larvae and product 4 also has reduced toxicity due to modified lactone ring.	Candida versatilis		Li et al. 2018b
	(3) C13H12O2	(4) C11H10O4
Peroxidase	C17H12O7			Recombinant Escherichia coli			Loi et al. 2020

Another important enzyme with AFB1 degradation properties, F₄₂₀H₂-dependent reductase, was isolated from Mycobacterium smegmatis (Taylor et al. 2010). This enzyme utilizes F₄₂₀H₂ as a cofactor and catalyzes the conversion of the α,β-unsaturated ester moiety in AFB1, leading to spontaneous hydrolysis (Figure 3a). Wu et al. (2009) studied the degradation of AFB1 by catalyzing the hydration of the terminal furan ring, which converts AFB1 into AFB2a using the enzymes of Aspergillus niger (Figure 3b). Afsharmanesh et al. (2018) expressed bacC, an oxidoreductase in Bacillus subtilis UTB1, and observed that the recombinant had antagonistic behavior against aflatoxin-producing Aspergillus flavus along with AFB1 degradative properties. It was observed that the expressed oxidoreductase exhibited significant sequence homology with one of the enzymes of Mycobacterium smegatis. Based on their observations and sequence similarity with the enzyme of Mycobacterium smegatis, a hypothetical degradation mechanism was established (Afsharmanesh et al. 2018). The degradation started from the bacC-mediated reduction of the double bond in the AFB1 lactone ring, and the subsequent hydrolysis of the ester bonds led to the formation of carboxylic acid, which could either be converted to aflatoxin D1 (AFD1) by decarboxylation or to aflatoxin D2 (AFD2) through the cleavage of the cyclopentenone ring (Afsharmanesh et al. 2018) (Figure 3c).

Figure 3. Proposed aflatoxin B1 degradation pathways. (a) F₄₂₀-mediated aflatoxin B1 conversion (Taylor et al. 2010); (b) Enzymatic aflatoxin B1 conversion into aflatoxin B2a by terminal furan ring hydration (Wu et al. 2009); and (c) Hypothetical aflatoxin B1 degradation scheme by oxidoreductase (Afsharmanesh et al. 2018).

A recent study demonstrated the AFB1 degradation potential of Bacillus subtilis catalyzed by AHL lactonase, which acts on the ester bond of the lactone ring (Pereyra, Martínez, and Cavaglieri 2019). Pereyra, Martínez, and Cavaglieri (2019) hypothesized that lactonase, along with other cellular and extracellular enzymes, is responsible for AFB1 degradation although they did not identify other essential enzymes responsible for the degradation. In addition, studies have shown bacteria- and fungi-mediated enzymatic AFB1 conversion, emphasizing the cleavage of the lactone ring as the principal mechanism of AFB1 degradation (Petchkongkaew et al. 2008; Samuel, Sivaramakrishna, and Mehta 2014). However, none of these studies have identified the enzymes responsible for AFB1 degradation.

Aflatoxin degradation products and their toxicity

AFB1 is converted to a highly reactive AFB1-8,9-epoxide by the cytochrome P₄₅₀ system, which can react with DNA (Zhang et al. 2016), subsequently inducing mutagenicity and/or carcinogenicity (Figure 4). The difuran and lactone rings are the major sites responsible for AFB1 toxicity, and destruction of these rings can lead to reduced toxicity (Vanhoutte, Audenaert, and De Gelder 2016). Aflatoxin-degrading enzymes can attack different locations of aflatoxin molecules. All enzymatic AFB1 degradation does not necessarily lead to the suppression of toxicity. Thus, it is critical to study the toxicity of the byproducts of enzyme-mediated aflatoxin biodegradation to ensure their safety for food applications. To date, only the degradation product AFB1-8,9-epoxide has been reported to be more toxic than AFB1 (Eshelli et al. 2015). The majority of enzymatic biodegradation ends up with some final metabolites, although complete mineralization is rarely observed. Rao et al. (2017) analyzed the degradation products of AFB1 using electron spray ionization-mass spectrometry and high-performance liquid chromatography but were unable to obtain any degradation products with m/z values of 313, 335, and 647, which are specific to AFB1. Similar results were observed by Sangare et al. (2014) and Farzaneh et al. (2012), who predicted that the degradation products would be chemically different from AFB1, indicating that AFB1 degradation is mediated by multiple enzymes present in the culture supernatant.

Figure 4. Production of aflatoxin B1 in food products, toxicity caused by the consumption of contaminated food products, and detoxification through enzymatic degradation.

Several degradation products such as aflatoxicol, AFB2a, AFD1, AFD2, phthalic anhydride, and AFB1-8,9-dihydrodiol have been reported. The aflatoxin degradation products depend on the type of enzyme and cleavage point of the aflatoxin molecule upon which enzymes act. For example, cleavage at the difuran ring, pentane group, and coumarin group produces AFB2a, aflatoxicol, and AFD1, respectively. Eshelli et al. (2015) and Samuel, Sivaramakrishna, and Mehta (2014) reported that Rhodococcus erythropolis and Pseudomonas putida could convert AFB1 to AFD1 and subsequently into AFD2, which do not contain cyclopentenone and lactone rings that are responsible for AFB1 toxicity. Recently, Taheur et al. (2020) studied the toxicity of AFB1 biodegradation products in vivo using a brine shrimp (Artemia salina) lethality assay and did not observe significant toxicity. Li et al. (2018b) applied salt-tolerant Candida versatilis CGMCC 3790 for AFB1 biodegradation and identified four nontoxic degradation products, with molecular formulas of C₁₄H₁₀O₄, C₁₄H₁₂O₃, C₁₃H₁₂O₂, and C₁₁H₁₀O₄. Li et al. (2018a) applied salt-tolerant Tetragenococcus halophilus CGMCC 3792 for enzyme-mediated AFB1 degradation and identified six nontoxic degradation products: C₁₄H₁₆O₂, C₁₄H₂₀O₂, C₁₄H₁₂O₃, C₁₄H₁₀O₄, C₁₆H₂₀O₄, and C₁₈H₁₆O₈. The studies conducted by Li et al. (2018a, 2018b) established that toxicity loss occurs due to the double bond removal in the terminal furan ring and destruction of the lactone ring (Table 1). Moreover, Iram et al. (2016) and Loi et al. (2017) observed reduced AFB1 toxicity upon modifying the lactone ring. Cell-free extracts, containing enzymes that can modify or cleave lactone and furan rings, are able to detoxify AFB1 (Afshar et al. 2020). An intermediate dihydro-hydroxy AFB1 (AFB2a: m/z = 330.07) was detected in the biodegradation products of laccase from Tremetes versicolor (Suresh et al. 2020). Although dihydro-hydroxy AFB1 is less mutagenic than AFB1, it has some hepatotoxic activity and can be spontaneously converted back to AFB1 (Rushing and Selim 2018). Suresh et al. (2020) identified eight degradation products; among them, three products were devoid of a lactone moiety and seven products had modified or completely cleaved furofuran. Recently, Loi et al. (2020) demonstrated that Rh_DypB converts AFB1 to AFQ1, which is significantly less toxic than AFB1. These studies indicate that biodegradation products are nontoxic or less carcinogenic to humans. Thus, microbial cell-free supernatants or microbial enzymes can be successfully applied to food products to control aflatoxin contamination.

Future genetic engineering approaches to eliminate toxic products of degradation

Common enzymatic AFB1 degradation products either have a lactone ring (e.g., AFQ1 and AFB2a) or furan ring (e.g., AFD1 and AFD2) or both (e.g., AFP1). Thus, these products have a certain degree of toxicity, even it is much lower than that of AFB1. The presence of such degradation products is of concern regarding safety toward humans. Therefore, their elimination is always desired to ensure safe and nontoxic food, although most current approaches are insufficient to achieve this goal. As a futuristic possible approach a chimeric enzyme with different types of laccases and reductases that simultaneously attack lactone and furan could be developed to address this. Such enzymes could target the furan and lactone ring in AFB1 to form a product free from furan and lactone rings. Furthermore, another group of recombinant enzyme containing dioxygenases and dehydrogenases will be constructed to attack the terminal ring of formed metabolites and open the ring structure to form linear nontoxic intermediates. These strategies are strongly supported by the findings of Xia et al. (2021), who developed a hybrid enzyme consisting of manganese peroxidase and zearalenone hydrolase for the simultaneous degradation of two toxins. If the proposed approach of the hybrid enzyme works efficiently, it would bypass the formation of toxic metabolites from AFB1 and could lead to the complete mineralization of AFB1.

Recent technological advancements for enzymatic aflatoxin degradation

Enzyme production by native strains is often associated with disadvantages such as low yield and low catalytic stability. Recent advances in enzyme engineering approaches, nanozyme technology, and bioinformatics provide solutions to improve enzyme yield and catalytic efficiency for effective aflatoxin degradation in food. The proposed recent technological advancements for aflatoxin degradation in food products are illustrated in Figure 4.

Enzyme engineering

The low catalytic activity of certain aflatoxin-degrading enzymes restricts their practical use in food. Moreover, the enzyme-mediated degradation of aflatoxin is often compromised in complex food systems because of the varied ranges of pH, ions, and salt content of food, which can interfere with enzymatic activity. Enzyme engineering has been successfully used to modulate the catalytic properties, stability, and production of enzymes for the degradation of aflatoxins in food. Different enzyme engineering techniques based on computation and site-directed mutagenesis have led to superior enzyme activity and stability. Yang et al. (2021) used in silico and site-directed mutagenesis approaches to modulate important catalytic amino acids to enhance the activity of Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D). Three amino acids present at the catalytic sites of TV-AFB1D, tyrosine 305 (Y305), glutamic acid (E436), and histidine (H554), were mutated to alanine (A) to generate four distinct mutants, namely H554A, Y305A/H554A, E436A/H554A, and Y305A/E436A/H554A. Among the four mutants, E436A/H554A showed the highest degradation efficacy, with 1.8-fold higher specific activity compared to wild-type TV-AFB1D (Yang et al. 2021). Li et al. (2019) fused thioredoxin to the N-terminus of MSMEG_5998 (an aflatoxin-degrading F₄₂₀H₂-dependent reductase from Mycobacterium smegmatis) and observed enhanced aflatoxin degradation by the recombinant enzyme (63%) compared to that for the native enzyme (31%). Another advantage of the genetic engineering approach is the utilization of fusion technology to produce hybrid enzymes capable of degrading two different mycotoxins simultaneously. Xia et al. (2021) used fusion technology to construct a hybrid enzyme using manganese peroxidase (PhcMnp) from Phanerochaete chrysosporium and zearalenone hydrolase (ZHD101 mutant V153H) from Clonostachys rosea with a “GGGGS” linker peptide. The fused enzyme simultaneously degraded aflatoxin and zearalenone. Such approaches are very useful for eliminating mycotoxins from foods that may be contaminated with different types of mycotoxins.

Nanozymes

Nanozymes are nanomaterials that can mimic enzymes to catalyze the conversion of a substrate to a product following the same kinetics and mechanisms under a given physiochemical condition. In 2007, Fe₃O₄ nanoparticles were discovered to have intrinsic peroxidase activity (Gao et al. 2007). Compared to native enzymes, nanozymes are associated with improved durability and stability. A nanohybrid MoS₂–Pt₇₄Ag₂₆ was developed that mimics the novel peroxidases and catalyzes the oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (Cai et al. 2016). A laccase-mimicking Cu/guanosine monophosphate nanozyme was prepared by mixing two common chemicals (Cu²⁺ and guanosine monophosphate) at room temperature (Liang et al. 2017). The Cu/guanosine monophosphate nanozyme was found to be stable for long storage times and under extreme temperature, pH, and salt conditions, being 2400-fold more cost-effective than commercial laccase. As laccases play a significant role in aflatoxin degradation, the Cu/guanosine monophosphate nanozyme may represent a potential candidate for aflatoxin removal from food. Other nanomaterials that mimic peroxidases have also been generated, such as CuMnO₂ nanoflakes (Chen et al. 2019), FeMnO₃ nanoparticle-filled polypyrrole nanotubes (Chi et al. 2018), and FePt nanoparticle-decorated graphene oxide nanosheets (Chen et al. 2018). Furthermore, Fe-based metal–organic frameworks have been developed and applied for AFB1 degradation, being able to degrade 85% of aflatoxin at an initial concentration of 50 ppm (Ren, Luo, and Wan 2019).

Bioinformatics approaches

With the advancement of bioinformatics approaches, various in silico tools have been developed to study enzyme structures and their catalytic activities. Laccase has been studied using in silico techniques, such as molecular docking and simulations. Homology modeling has been performed to predict the three-dimensional structure of laccase (Dellafiora et al. 2017); determination of the three-dimensional structure of enzymes can help to predict the possible mechanisms of binding between the substrate (aflatoxin) and the enzyme. Molecular docking predicted that laccase interacts with aflatoxin close to the T1 copper center of the enzyme (Liu, Mao et al. 2020). A homology model was also developed, and molecular docking was performed to predict the molecular-level interactions between the enzyme and substrate (AFB1, AFB2, AFG1, and AFG2) (Liu, Mao et al. 2020). Such analyses provide an understanding of the molecular basis of enzyme–substrate interactions, differences among different aflatoxin enzyme surface and ligand interactions, and specific interactions at the catalytic sites of enzymes. The in silico results can thus help broaden our understanding of the molecular mechanisms of degradation, providing a basis for future molecular modifications of aflatoxin-degrading enzymes such as laccase. Moreover, in silico studies provide information for the selection of an effective enzyme to control aflatoxins in fermented foods. Without these techniques, it can be laborious to screen every enzyme for its effectiveness under laboratory conditions. In silico approaches are thus effective analytical tools for investigating enzyme–aflatoxin interactions correlated with the binding affinity.

Statistical optimization

Enzymatic reactions are highly influenced by reaction parameters such as temperature, pH, co-factors, metal ions, and substrate concentrations. Many studies have shown a significant increase in catalytic activity following the optimization of all reaction parameters. There are several approaches such as the “one factor variable at a time” approach, response surface methodology, Taguchi design, Plackett–Burman design, genetic algorithms, and artificial neural networks to optimize reaction parameters to achieve the maximum catalytic efficiency (Kumar, Chhabra, and Shukla 2017). These approaches may be used to improve the enzymatic degradation of aflatoxins in fermented foods. Some researchers have applied the “one factor variable at a time” approach and response surface methodology to maximize enzymatic aflatoxin degradation (Eshelli et al. 2015; Subramanian et al. 2017). The reaction conditions for three actinomycete species, Streptomyces lividans TK 24, Rhodococcus erythropolis ATCC 4277, and Streptomyces aureofaciens ATCC 10762, were optimized for the maximum biodegradation of AFB1, and the optimal conditions for Streptomyces lividans and Streptomyces aureofaciens were 30 °C and pH 5, whereas those for Rhodococcus erythropolis were 30 °C and pH 6 (Eshelli et al. 2015). The thermal degradation of aflatoxin-spiked potato dextrose broth was optimized for parameters such as temperature (110–119 °C), pH (4–10), and time (10–20 min) using a quadratic model (Subramanian et al. 2017). A combination of the Plackett–Burman design and Box–Behnken design was applied to optimize Panus neostrigosus culture conditions to obtain culture filtrates with maximum aflatoxin degradation abilities. Fifteen variables were selected for the Plackett–Burman design, and three were selected for the Box–Behnken design. Maximum aflatoxin degradation (49%) was obtained from the culture filtrate produced under optimal growth conditions (Söylemez, Yamaç, and Yıldız 2020).

Microbial consortium for aflatoxin degradation

Complete aflatoxin detoxification requires several enzymes. These enzymes may be produced by either a single strain or by multiple strains of microorganisms. Microbial consortium technology has become increasingly popular for the complete degradation of several complex organic compounds (Wang et al. 2016). Microbial consortia have certain advantages over a single microbial strain for the complete degradation of complex organic compounds (Yuan et al. 2016). Different types of microorganisms are responsible for multiple enzyme activities and the varied depolymerization of complex compounds. Owing to a variety of microorganisms with different enzymatic profiles, microbial consortia can perform more complex tasks and tolerate different environments better than a single strain (Bhatia et al. 2018). Both aerobic and anaerobic bacteria can degrade AFB1 (Salati et al. 2014), and microbial degradation is facilitated by the co-operation of anaerobic and aerobic bacteria (Kato et al. 2005). Wang, Zhao et al. (2017) applied a thermophilic microbial consortium TADC7 for AFB1 degradation and observed more than 95% AFB1 degradation within 72 h at an optimal temperature of 55–60 °C. The microbial consortium was able to tolerate AFB1 doses of up to 5000 µg/L without any inhibitory effect on aflatoxin degradation, and the predominant microorganisms, Geobacillus and Tepidimicrobium, were identified to be responsible for AFB1 degradation (Wang, Zhao et al. 2017). Wang, Zhao et al. (2017) applied a thermophilic microbial consortium for aflatoxin degradation in food and feed products. A study was conducted recently on the kombucha (a fermented black tea) microbial consortium, suggesting that this consortium comprises several AFB1-degrading microorganisms (Taheur et al. 2020). Yeast and lactic acid bacteria isolated from kombucha show AFB1 degradation capabilities (Taheur et al. 2020). Taheur et al. (2020) observed 97% AFB1 degradation by the kombucha microbial consortium, similar to the degradation (95%) observed by Wang, Zhao et al. (2017). However, significantly different degradation times were observed in the two studies; Taheur et al. (2020) observed maximum degradation on the 7^th day, whereas Wang, Zhao et al. (2017) observed maximum degradation on the 3^rd day. The difference in the degradation duration depends on the composition, type of microorganisms, and enzymes produced by the microorganisms in the consortium. From kombucha, Taheur et al. (2020) identified Candida sorboxylosa KOP10, Hanseniaspora opuntiae KOT8, and Pichia occidentalis KOP7 to be responsible for AFB1 degradation. Among them, Pichia occidentalis KOP7 was found to be the most potent for AFB1 degradation. Wang, Zhao et al. (2017) extended their research on the microbial consortium TADC7 for the simultaneous degradation of multiple mycotoxins and used the cell-free supernatant to achieve 93.8% AFB1 and 90.3% zearalenone degradation at 72 h. Taheur et al. (2017) also used the kefir grain microbial consortium to detoxify AFB1, zearalenone, and ochratoxin A, and achieved 80–100% detoxification. The mechanism of detoxification by kefir grain microorganisms was binding and/or adsorption rather than enzyme reaction, by Acetobacter syzygii, Kazachstania servazzii, and Lactobacillus kefiri (Taheur et al. 2017). Although few reports have shown the efficiency of microbial consortia for aflatoxin degradation, microbial consortia could be an effective tool for controlling the quantities of toxins in fermented foods and other food products.

Risk assessment of aflatoxin in fermented food

Fermented foods are an integral part of diets worldwide. It is predicted that the global market for fermented food products and ingredients may reach up to $689.34 billion by 2023 (Research and Markets 2021). However, fermented food industries face several challenges such as contamination by pathogenic bacteria, contamination by toxins and toxin-producing microorganisms, and contamination with pesticides and heavy metals. The fermentation process enhances the flavor, taste, and shelf life of fermented food products, and fermenting microorganisms can release a number of bioactive compounds such as exopolysaccharides, peptides, vitamins, oligosaccharides, and neurotransmitters. However, several unwanted toxic contaminants such as mycotoxins, biogenic amines, and bacterial toxins can also be released into fermented food products. The presence of a high amount of undesirable toxic compounds in fermented foods beyond safe limits can lead to serious health risks for consumers.

Cereals and legumes are the raw materials used for preparation most of fermented food products, such as fermented soybean paste (doenjang), red pepper paste (gochujang), African oil bean (ugba), locust beans (iru), maize gruel (ogi), maize meal (mahewu), rice, barley, and/or soybean paste (miso), and bean paste (tương). Cereals and legumes contaminated with toxicogenic fungi during the pre-harvest or post-harvest periods can lead to toxin contamination in the final fermented food product. Once mycotoxins contaminate food products, it is exceedingly difficult to safely remove them from food products. Thus, it is almost certain that humans will be exposed to mycotoxins if food products are contaminated. Sivamaruthi, Kesika, and Chaiyasut (2018) reported that various fermented food products and raw materials worldwide were found to be contaminated with aflatoxins. For example, soy fermented products, tương (Thanh and Viet Anh 2016) and thua-nao (Petchkongkaew et al. 2008), South African alcoholic beverages, Nigerian foods (iru and ogiri), and fermented soybean (meju) (Sivamaruthi, Kesika, and Chaiyasut 2018) were found to be contaminated with aflatoxin. Therefore, it is desirable to control aflatoxins in fermented foods, and researchers are continuously developing aflatoxin degradation techniques. Aflatoxin biodegradation is accepted as a relatively safe and attractive option to eliminate aflatoxins from fermented foods (Petchkongkaew et al. 2008). Several microorganisms isolated from different ecological niches, including fermented food products, can be used for aflatoxin biodegradation. These microorganisms and their products can be directly applied to the food fermentation process without many regulatory issues, as they have been isolated from food products that have been extensively consumed by humans for centuries. Reports on the application of microorganisms isolated from fermented foods for aflatoxin degradation/detoxification are listed in Table 2.

Table 2. Aflatoxin detoxifying microorganisms isolated from fermented food products.

Fermented food	Microorganism isolated/identified	Reduction of aflatoxin (%)	Reduction mechanism	Application to control aflatoxins in foods	References
Kefir grains	Lactobacillus kefiri, Kazachstania servazzii, Acetobacter syzygii	82–100	Adsorption	To develop fermented dairy products with new probiotic characteristic and adsorption of mycotoxins	Taheur et al. 2017
Kombucha beverage	Pichia occidentalis, Candida sorboxylosa, Hanseniaspora opuntiae	97	Adsorption and enzymatic degradation	Aflatoxin detoxification in food and feed industry	Taheur et al. 2020
Iru fermented locust beans	Bacillus subtilis		Enzymatic degradation by extracellular fraction	Application as an effective soaking agent to reduce the aflatoxin B1 in foods and feeds materials	Watanakij, Visessanguan, and Petchkongkaew 2020
Soy sauce mash	Candida versatilis	41.23	Adsorption and enzymatic degradation	To remove aflatoxins from fermented foods as well as flavor enhancing agents	Li et al. 2018b
Fermented chinese soybean	Aspergillus niger	98.65	Glutathione- mediated biotransformation	Potential for application for detoxification of aflatoxin B1 in food and feed products	Qiu et al. 2021
Soy sauce mash	Tetragenococcus halophilus	91.99	Adsorption proceeded by enzymatic degradation	To remove aflatoxins from fermented foods as well as flavor enhancing agents	Li et al. 2018a
Chinese fermented soybean	Aspergillus niger	85.8	Inhibition of mycelia growth, spore germination, and sporulation of Aspergillus flavus together with degradation of aflatoxin B1 with reduced mutagenicity and toxicity	Filtrate is economic and effective agent that can be applied in fermented food to prevent toxigenic Aspergillus sp. and aflatoxin B1 contamination.	Xu et al. 2013
Thai fermented soybean (Thua-nao)	Bacillus licheniformis, Bacillus subtilis	74–85	Unknown	Suggested to control the quality of fermented soybean	Petchkongkaew et al. 2008
Fermented maize dough (Kenkey) and sorghum beer (Pito)	Saccharomyces cerevisiae		Binding	Recommended as starter cultures for relevant indigenous fermented foods where high aflatoxin level is a potential health risk	Shetty, Hald, and Jespersen 2007

The mechanism of action for aflatoxin-detoxifying microorganisms may involve adsorption or enzyme-mediated degradation (Table 2). The enzymatic degradation is more advantageous process due to the irreversibility of the enzyme catalyzed reaction where most of the degraded products showed nontoxicity in in vivo studies (Table 1); therefore, it is safe to apply enzymes to control aflatoxin levels in food. Li et al. (2018a, 2018b) identified two salt-tolerant microbial strains from soy sauce mash, Candida versatilis CGMCC 3790 and Tetragenococcus halophilus CGMCC 3792, which were able to enzymatically degrade aflatoxins. These isolated microbial strains exhibited flavor-enhancing properties, which is an additional advantage for improving the quality of food products.

Techno-economic feasibility of aflatoxin degradation in the fermented food industry

The successful implementation of aflatoxin decontamination technology in the fermented food industry should meet the following criteria: (1) it should be effective at reducing the aflatoxin levels below safe limits for consumption; (2) it should not change the nutritional quality and sensory properties of fermented foods; (3) it should be cost effective, time efficient, and eco-friendly; (4) no toxic effect should be produced by the decontamination strategy; and (5) it should be applicable to diverse food products. In processed and fermented foods, biological methods utilizing whole microorganisms and aflatoxin-degrading enzymes can be effectively applied for aflatoxin management (Marshall et al. 2020; Wu et al. 2009). Owing to the high specificity, enzymes do not interact with other food components, thereby preserving the original food quality without altering the essential organoleptic and nutritional properties (Wu et al. 2009). Moreover, enzymes can be utilized in the immobilized form, thus ensuring continued use at the industrial level. Another notable advantage of utilizing enzymes is their energy-efficient and environment-friendly nature (Wu et al. 2009). Though enzymatic degradation meets most of the requirements for industrial commercialization, but still suffers from economic and technical issues. One of the major limitations is the relatively high cost of aflatoxin-degrading enzymes owing to the low production yields of these enzymes from their native host. The production of aflatoxin-degrading enzymes using wild strains and standard fermentation procedures is not economically feasible for large-scale industrial applications. Furthermore, enzymatic degradation takes place at an optimum temperature and pH, and could be inhibited or activated by other reagents and metal ions. Fermented food is a complex matrix with many metabolites and varied pH levels, so it is difficult to efficiently degrade aflatoxins in such complex environments using native enzymes that are not stable at a wide range of pH and temperature. Several recent technological advancements that improve enzyme yield, activity, and stability, making it economically and technologically feasible for the fermented food industry, have already been discussed in the previous section. Furthermore, specialized equipment and technically skilled labor are required, which may impose an additional burden on the overall production cost.

Conclusion and future aspects

Biodegradation mediated by microorganisms and enzymes is emerging as a promising technique owing to its economical, efficient, and relatively safe nature. The results summarized in the current review focused on the microorganism- and enzyme-mediated degradation of aflatoxin and linked the degradation process with application in food. Laccases, peroxidases, reductases, and lactonases are the main enzymes involved in aflatoxin degradation, mainly targeting difuran or lactone rings, which are primarily responsible for aflatoxin toxicity. Protein engineering approaches, such as site directed mutation and fusion of proteins, have emerged as efficient tools to improve the efficiency of aflatoxin-degrading enzymes. Consistent to this, nanozymes are also gaining acceptability due to their higher efficiency and rapid pace to degrade aflatoxin. Moreover, bioinformatics approaches help to elucidate the structure of aflatoxin-degrading enzymes to predict catalytic improvements and the favorability of enzyme–aflatoxin interactions. The microbial consortium approach is a promising for complete aflatoxin degradation because it allows various microorganisms that possess different types of enzymes to enhance aflatoxin degradation.

Many aflatoxin-degrading microorganisms have been isolated from fermented foods. Most of these aflatoxin-degrading microorganisms have been tested in vitro, but their direct application in the food has not yet been extensively studied; therefore, further research is required to develop an effective mechanism for controlling aflatoxin contamination. In the future, efficient microorganisms capable of degrading aflatoxin need to be isolated and utilized as starter cultures to ensure the aflatoxin-free preparation of fermented food. Moreover, appropriate mechanisms for enzymatic aflatoxin degradation has not been sufficiently studied, and knowledge on most degradation pathways is fragmented. Furthermore, it is essential to understand the mechanisms of aflatoxin detoxification using in vivo studies. The exploration of novel genes for aflatoxin degradation enzymes using metagenomics is a future direction in this field. Enzyme engineering approaches could also be applied to increase the operational stability of enzymes during food processing, in addition to improve the degradation efficiency. As a core message, the review advocates the use of aflatoxin-degrading microorganisms by fermented food industries for the operative control of aflatoxin. Furthermore, the use of aflatoxin-degrading enzymes is recommended to counter aflatoxin because of the high specificity of enzymes and their negligible effect on food sensory qualities. However, the toxicity of the aflatoxin degradation products must be evaluated before the application of these enzymes in the food industry.

Author contributions

VK, J-JS, and MK: conceptualization, writing, reviewing, and editing; VK, AB, SR, and GD: data curation, writing, and original draft preparation.

Declaration of interest

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by Yeungnam University research grant in 2020, the National Research Foundation (NRF) of Republic of Korea under the frameworks of the Priority Research Centers Program (NRF-2014R1A6A1031189), and the NRF grant funded by the Korea government (MSIT) (NRF-2021R1A2C2094641).