toxins Review Aflatoxin Detoxification Using Microorganisms and Enzymes Yun Guan 1, Jia Chen 1, Eugenie Nepovimova 2, Miao Long 1,* , Wenda Wu 2,3,* and Kamil Kuca 2,* 1 Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China; [email protected] (Y.G.); [email protected] (J.C.) 2 Department of Chemistry, Faculty of Science, University of Hradec Kralove, 50003 Hradec Kralove, Czech Republic; [email protected] 3 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China * Correspondence: [email protected] (M.L.); [email protected] (W.W.); [email protected] (K.K.) Abstract: Mycotoxin contamination causes significant economic loss to food and feed industries and seriously threatens human health. Aflatoxins (AFs) are one of the most harmful mycotoxins, which are produced by Aspergillus flavus, Aspergillus parasiticus, and other fungi that are commonly found in the production and preservation of grain and feed. AFs can cause harm to animal and human health due to their toxic (carcinogenic, teratogenic, and mutagenic) effects. How to remove AF has become a major problem: biological methods cause no contamination, have high specificity, and work at high temperature, affording environmental protection. In the present research, microorganisms with detoxification effects researched in recent years are reviewed, the detoxification mechanism of microbes on AFs, the safety of degrading enzymes and reaction products formed in the degradation process, and the application of microorganisms as detoxification strategies for AFs were investigated. One of the main aims of the work is to provide a reliable reference strategy for biological detoxification of AFs. Keywords: aflatoxin; biological detoxification; detoxification mechanism; degradation products; probiotics Citation: Guan, Y.; Chen, J.; Key Contribution: The mechanism, advantages and disadvantages of microorganisms and enzymes Nepovimova, E.; Long, M.; Wu, W.; to detoxification of aflatoxins are reviewed; A reliable reference strategy for biological detoxification Kuca, K. Aflatoxin Detoxification of aflatoxins is provided. Using Microorganisms and Enzymes. Toxins 2021, 13, 46. https://doi.org/ 10.3390/toxins13010046 1. Introduction Received: 13 December 2020 Accepted: 6 January 2021 Mycotoxins are metabolites of fungi that are ubiquitous in cereal crops and animal Published: 9 January 2021 forage [1]. One group of well-known mycotoxins, aflatoxins (AFs), are secondary metabo- lites produced mainly by Aspergillus flavus, which produces both aflatoxin B1 (AFB1) and Publisher’s Note: MDPI stays neu- aflatoxin B2 (AFB2), and by Aspergillus parasiticus, which produces aflatoxin G1 (AFG1) and tral with regard to jurisdictional clai- aflatoxin G2 (AFG2)[2]. They have a high degree of hepatotoxicity, nephrotoxicity, and ms in published maps and institutio- immunotoxicity [3]. Among them, AFB1 is the most toxic and is well known for its toxic nal affiliations. carcinogenic and teratogenic mutation effects [4,5]. As a result, it was categorized as a Class I carcinogen by the World Health Organization in 1993 [6,7]. The long-term consumption of food contaminated with AFs can induce inflammatory damage to hepatocytes [8]. Furthermore, the AF-DNA adducts can result in the production Copyright: © 2021 by the authors. Li- of cancer cells [9], leading to liver cancer [10,11]. In addition, AFB1 can induce the apoptosis censee MDPI, Basel, Switzerland. of CASP3 and BAX, and shows extensive cytotoxicity to neuronal cells, including ROS This article is an open access article accumulation, DNA damage, S-phase arrest, and apoptosis [12]. AFs can also destroy distributed under the terms and con- the metabolic pathways of a variety of intestinal flora. This may affect energy supply ditions of the Creative Commons At- and lead to certain metabolic diseases [13,14]. Today, South-East Asia remains a high-risk tribution (CC BY) license (https:// creativecommons.org/licenses/by/ area for acute AF poisoning [15]. Molecular structures of four naturally occurring AFs are 4.0/). illustrated in Figure1. Toxins 2021, 13, 46. https://doi.org/10.3390/toxins13010046 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 19 Toxins 2021, 13, 46 2 of 17 remains a high-risk area for acute AF poisoning [15]. Molecular structures of four natu- rally occurring AFs are illustrated in Figure 1. AFB1 AFB2 AFG1 AFG2 Figure 1. Structures of someFigure natural 1. Structures AFs of some (Aflatoxins natural AFs (Aflatoxins B1 and B G1 and1 haveG1 have double bonds bonds at positions at positions8–9; aflatoxins 8–9;B2 and aflatoxinsG2 do B2 and G2 do not). not). AFs are oftenAFs are detected often detected in grains,in grains, nuts, and and spices spices [16,17]. Contamination [16,17]. Contamination occurs occurs readily when feed andreadily food when feed are and exposed food are exposed to highto high temperature temperature and high humidity and high [18]. The humidity [18]. The toxic toxic effects of AFs are not only manifested in feeding. Animals that consume contami- effects of AFsnated are feed not are likely only to manifestedbe poisoned [19]. However, in feeding. the toxins Animals found in the that animal consume contaminated by-products (e.g., milk and milk products) will enter other animals in the food chain, feed are likelywhich to can be result poisoned in further serious [19]. conseq However,uences and thespread toxins the contamination found more in the animal by-products (e.g., milk andwidely milk [20]. Finding products) ways to safely will and enter efficiently other detoxify animals food has thus in become the food a focus chain, which can result of research [21]. Contamination of AFs in food and feed samples in some countries is in further seriousdisplayed consequences in Table 1. and spread the contamination more widely [20]. Finding ways to safely andTable 1. efficiently Contamination of detoxifyAFs in food and feed. food has thus become a focus of research [21]. Contamination of AFs in foodRate of andCon- feed samples in some countries is displayed in Table1. Locality Sample AFs Toxin Level a Refs tamination (%) Fish feed in the Table 1. 48 <40 µg/kg Uganda Contaminationfactory of AFs in food and feed. B1 [22] Lake Victoria Basin Fish feed in the 63 >400 µg/kg farm Rate of a Locality Sample Groundnut AFs Toxin Level Refs Contamination (%) 84.7 µg/kg Uganda seeds 81 — b [23] Multiple districts Milled ground- Uganda Fish feed in the factory 48 1277.5 µg/kg <40 µg/kg nuts B1 [22] Lake Victoria Basin Fish feed in the farm 63 >400 µg/kg Uganda Groundnut seeds 84.7 µg/kg 81 — b [23] Multiple districts Milled groundnuts 1277.5 µg/kg Cameroon Catfish 100 B1 31.38 ± 0.29 ppb [24] B1 105.4 µg/kg Nigeria B2 6.92 µg/kg Dried beef meat (as sold) 66 [25] Ekiti State G1 40.49 µg/kg G2 2.60 µg/kg 20 B1 0.1 µg/kg Mexico Oaxaca-type cheese 30 G 0.6 µg/kg [26] Mexico City (as sold) 1 57 M1 1.7 µg/kg Raw peanuts 12.8–537.1 µg/kg Malaysia —— [27] Peanut sauce 5.1–59.5 µg/kg Sri Lanka Corn 63.33 60–70 ppb B1 [28] Anuradhapura Corn-growing soil 90 350–400 ppb India Cereals in the family 82 B >1µg/ kg [29] Mahabubnagar 1 Thailand Sesame (as sold) 9 — >2 µg/kg [30] a Unsigned data represent the average rate of contamination. b This symbol indicates unknown or not mentioned. Toxins 2021, 13, 46 3 of 17 AFs can be detoxified using physical, chemical, and biological detoxification methods, and a great deal of research has been carried out using these methods in the past few decades [31,32]. Physical methods are those most commonly used; for example, adsorbents are employed to undertake physical adsorption to control toxin contamination [33]. Al- though adsorbent products can reduce the bioavailability of mycotoxins, in practice, the toxins cannot be completely adsorbed [34]. In recent years, after continuous improvement, nanotechnology has been applied to adsorbents, such as magnetic adsorbents, whose adsorption capacity has been much improved [35]. However, physical methods show many disadvantages, e.g., limited applicability, poor detoxification effect, and limited detox product status [36]. Chemical methods involve treatment with acid, alkali, or ox- idizing agent [37]. The use of chemical substances such as chlorine dioxide to disinfect toxins [38] may impair the appearance and taste of food. After chemical treatment, chemical residues in food may be harmful to humans [39]. Neither approach is the better option for detoxification. Biological detoxification also has certain drawbacks, such as the diffi- culty of controlling microbial performance and the safety of the newly formed product to the body [39]; however, biological detoxification has high specificity, produces harmless products, and can even completely detoxify samples under appropriate conditions [37,40]. Thus, biological detoxification is gradually becoming the most suitable detoxification approach [41,42]. Beneficial intestinal bacteria have many important functions. They produce various nutrients for the host, prevent infections caused by intestinal pathogens, and regulate the immune response [43]. At the same time, the life activity metabolites of microorganisms (such as exogenous antioxidant compounds) can induce activity among genes related to the oxidative stress toxicity of AFs, restore the oxidative balance destroyed by mycotoxins, and prevent the production of ROS and RNS [44]. Therefore, the use of microorganisms to detoxify AFs is a promising new technology with broad application prospects; as such, their use is a research hotspot both for the beneficial effects and AF detoxification [41,45]. 2. Microorganisms with Detoxification Effects Different microorganisms exert detoxification effects toward AFs [46]. The microor- ganisms that exert detoxification effects on AFs are listed in Table2. Table 2.