Predominant Mycotoxins, Pathogenesis, Control Measures, and Detection Methods in Fermented Pastes

Fermented pastes are some of the most popular traditional products in China. Many studies reported a strong possibility that fermented pastes promote exposure to mycotoxins, including aflatoxins, ochratoxins, and cereulide, which were proven to be carcinogenic and neurotoxic to humans. The primary mechanism of pathogenicity is by inhibiting protein synthesis and inducing oxidative stress using cytochrome P450 (CYP) enzymes. The level of mycotoxin production is dependent on the pre-harvest or post-harvest stage. It is possible to implement methods to control mycotoxins by using appropriate antagonistic microorganisms, such as Aspergillus niger, Lactobacillus plantarum, and Saccharomyces cerevisiae isolated from ordinary foods. Also, drying products as soon as possible to avoid condensation or moisture absorption in order to reduce the water activity to lower than 0.82 during storage is also effective. Furthermore, organic acid treatment during the soaking process reduces toxins by more than 90%. Some novel detection technologies based on magnetic adsorption, aptamer probes, and molecular-based methods were applied to rapidly and accurately detect mycotoxins in fermented pastes.


Introduction
Fermented pastes are very popular in China and include soybean paste, thick broad-bean paste, flour paste, and chili paste. The unique flavor, taste, and nutritional components of the paste products mainly depend on the long-term fermentation by aroma-producing microorganisms [1]. The antioxidant activities of fermented pastes may be improved through prolonged fermentation [2]. However, several undesired mycotoxins may exist and can lead to human disease due to the high risk of paste contamination during the long-term fermentation process [3]. Mycotoxins produced by fungi play the most significant role in food contamination [4]. For instance, high levels of mycotoxins were detected in wheat, coffee, grape products, and even in animal feed [5]. Some fermented products were reported to contain an abundance of Aspergillus, Penicillium, and Fusarium spp., which are correlated with the presence of aflatoxins or ochratoxin A [6]. The production of aflatoxins during paste fermentation is affected by fermentation conditions, including the protein ratio of raw materials, starter cultures, and so on [24]. A. flavus and its close relatives are commensal with the crop and infect it during growth to produce aflatoxins before harvest. Some saprophytic species contaminate crops only after harvest [25]. Previous researchers showed that the growth conditions of these saprophytic species were above 37 • C and close to 0.80 water activity [26].
Crops contaminated by different Aspergillus species demonstrate various levels of risk regarding the presence of aflatoxins in final fermented pastes. For example, small grains like soybean and wheat do not have affinity with Aspergillus species, and aflatoxins can only be produced during untimely drying or storage at high-temperatures after harvest [27]. Crops such as peanuts and maize have higher risk levels than others, because A. flavus and A. parasiticus are commensal with these crops [28]. Contaminated soil further promotes mold on the plants if unharvested grains are not cleaned up in time [29]. Aflatoxins are not be degraded during the fermented process [30].

Ochratoxins
Ochratoxins are secondary metabolites produced by Penicillium or Aspergillus fungi under diverse conditions, with 18 related isomers, of which ochratoxin A (OTA) is the most toxic and considered to be teratogenic, neurotoxic, nephrotoxic, and carcinogenic in humans [31,32]. OTA contamination occurs in various foods, including soybeans, dried fruits, and coffee, and was also detected in fermented wines and grape juices [33]. Furthermore, the biosynthetic pathway of OTA in fungi was confirmed to be strongly related to polyketide synthase (otapksPN) and non-ribosomal peptide synthetase (npsPN), which are regarded as the primary enzymes [34]. Previous research demonstrated that there were three steps of OTA biosynthesis, namely, polyketide synthesis, acyl activation, and de-esterification by esterase [35].
The occurrence of OTA in fermented pastes is influenced by many factors, including the type of crop, the farming practice, the machining process, and environmental conditions. Previous studies showed that the prevalence of OTA in maize was very limited compared with other crops [36]. OTA was detected in 34.2% of wheat samples in six provinces of China [37]. Unlike aflatoxins, ochratoxin-producing pathogens are mostly regarded as post-harvest spoilage. For instance, Penicillium species were confirmed to be strongly correlated with stored cereals, such as soybeans, beans, and green coffee [38], therefore, the storage and process conditions were found to greatly influence the final products. Moisture is also a critical factor in the contamination of OTA during storage; the safe-storage water activity level is below 0.7 [39] and the risk of OTA occurrence is increased if the drying process is not rapid and moisture is absorbed on the surface of products in cold climates. Damage to the raw materials during harvest, processing, and storage is also a significant factor [40].

Cereulide
Bacillus cereus, a spore-forming, poisonous, Gram-positive bacteria, is especially relevant for immunocompromised patients, drug users, newborns, and post-surgical patients. Strains isolated from parenteral infections are able to synthesize necrotizing exotoxin-like phospholipase and hemolysin [41]. B. cereus can be isolated from human dentin plaques, but accounts for a small proportion of oral infections. Collegenase-producing Soc 67 and Ply 19 were isolated from plaque in a patient with gingivitis and a teenager with periodontitis [42].
Bacillus cereus can be isolated from a variety of fermented foods and environments, such as doenjang and soybean paste. B. cereus phages were isolated from 47 traditional Korean fermented foods, as well as 63.6% of doenjang samples, 100% of cheonggukjang samples, and 40% of gochujang samples [43]. Cereulide is considered to be an important foodborne toxin during paste fermentation due to its ability to cause diarrheal (heat-labile) and emetic (heat-stable) food poisoning after ingestion of more than 10 4 Colony-Forming Units (CFU) per gram of food [44,45]. Controlling cereulide in in industrial practices is difficult due to the wide range of temperature (5-55 • C) in which B. cereus can grow [46].

Cancer Diseases
A large amount of research regarding the toxicological mechanisms of mycotoxins revealed that many foodborne mycotoxins inhibit protein synthesis and induce oxidative stress, thereby causing various cancers [47]. For instance, fermented pastes contaminated by AFB 1 cause latent damage, leading to human hepatocellular carcinoma (HCC), a common liver cancer. Previous research confirmed that the metabolic activation of AFB 1 had a strong correlation with cytochrome P450 (CYP) enzymes, including CYP 1 A 2 , and CYP 3 A 4 , which are mainly expressed in the human liver [48]. HCC is metabolized to an AFB 1 -epoxide, and then adducts into AFB 1 -DNA [49], induces the transversion of G to T within codon 249 of the tumor suppressor gene p53, which is associated with liver cancer [9].
Furthermore, OTA was reported to be a group 2B human carcinogen by the International Agency for Research on Cancer (IARC) [32]. Due to the nephrotoxicity of OTA, ingesting products abundant in OTA increases the risk of suffering from Balkan Endemic Nephropathy (BEN) and Tunisian Nephropathy (TCIN) [50]. Recent research revealed that oxidative stress induced by OTA in HK-2 cells regulated the translocation of the transcription factors aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) [51]. Activated AhR leads to immunosuppression and cancer by regulating CYP 1 A 1 and CYP 1 A 2 enzymes, and activated PXR has a relationship with CYP 3 A 4 , playing a role in phase I metabolism. Furthermore, activated Nrf2 inhibits cell death and decreases the expression of AhR and PXR in the presence of OTA, resulting in reduced renal damage [52]. Therefore, the regulation of transcription factors Nrf2, PXR, and AhR could help to treat and prevent renal injury by OTA ( Figure 2) [53].

Neurodegenerative Diseases
Alongside carcinogenicity, OTA was demonstrated to impact migration, neuronal proliferation, and DNA content, thereby leading to neurodegenerative diseases and brain dysfunction of human such as parkinsonism and Alzheimer's disease [54]. The neurotoxicity of the hippocampus, ventral mesencephalon, and striatum is more pronounced than in the cerebellum [55]. The mechanism of neurotoxicity involves OTA causing acute depletion of striatal dopamine and decreasing the immunoreactivity of striatum tyrosine hydroxylase, alongside a transient inhibition of DNA oxidative repair activity (oxyguanosine glycosylase, OGG 1 ) [56]. Furthermore, OTA treatment inhibits the activation of SH-SY5Y, caspase-9, and caspase-3, which is accompanied by a decrease in mitochondrial membrane potential [57].

Emetic Illness
The emetic type of foodborne illness is induced by the small, cyclic, heat-stable toxin cereulide, which causes vomiting and nausea. [63]. The ces gene encodes cereulide synthetase, which is associated with the emetic toxin-producing strain of B. cereus [64]. Cereulide destroys the electrochemical gradient of membranes and inhibits fatty acid metabolism in mammalian cells by uncoupling mitochondrial ATP synthesis and disrupting the mitochondrial membrane potential [65]. Furthermore, the biological activity of the cereulide toxin impairs mitochondrial functionality (Figure 3) [66].
The pathogenicity mechanism of mycotoxins is briefly listed in Table 2.

Biocontrol Method
Biocontrol is one of the most effective techniques of controlling various mycotoxins; it uses safe microorganisms in fermented pastes to inhibit pathogen growth [67]. The advantage of the biocontrol method is that the growing conditions of related antagonistic microorganisms are simple enough, and they have strong abilities to multiply in foods for long periods of time [68]. In addition, unlike antagonists, they do not carry the risk of producing allergenic spores, which is harmful to the environment and human health [69].
Several works showed that many lactic acid bacteria (LAB) strains were able to restrict the growth of A. flavus. For instance, both L. plantarum and related low-molecular-weight substances showed significant antifungal activity [70]. Recent research revealed that various novel metabolites of LAB, including 13-hydroxy-9-octadecenoic acid, 10-hydroxy-12-octadecenoic acid [71], phenolic antioxidants [72], and spermine-like and short cyclic polylactates [73], showed lower minimum inhibitory concentrations of mycotoxins compared to the ordinary organic acids. Lactobacillus fermentum YML014 was also shown to reduce A. flavus by up to 50% after isolation from Nigerian fermented food (Cassava) [74].
The mechanism of LAB against fungal mycotoxins involves metabolic conversion by binding the related mycotoxin to the cell wall through adsorption and enzymatic degradation [75]. The coexistence of biocontrol agents and mycotoxins in the environment result in competition for nutrients and space, thereby directly impacting the growth of various mycotoxins. In addition, some biocontrol strains modify the external environment to inhibit mycotoxin production. For instance, LAB-induced decrease in pH led to a reduction in aflatoxin production by 91% by decreasing the stability of related mycotoxins [76]. Furthermore, oxidative stress induced by LAB was also responsible for the production of mycotoxins. It was reported that aflatoxins were produced under oxidative stress conditions, so antioxidants could play a restoration role [77]. The other widely accepted mechanism is that mycotoxins bind to LAB cell wall compounds through absorption. Polysaccharides and peptidoglycans are regarded as the most important components to bind OTA to L. plantarum and Lactobacillus sanfranciscensis, which could be enhanced through genetic modification [78]. Unlike OTA, aflatoxins were demonstrated to bind peptidoglycans and teichoic acids of the Lactobacillus rhamnosus cell wall; this binding ability was decreased by heat-treating [79].
Moreover, the mechanism of mycotoxin regulation by Aspergillus niger FS10 was also studied. The results confirmed that the culture filtrate of A. niger FS10 had a strong ability to disrupt spore morphology, deform the cell wall, and restrict spore germination [80]. Also, the degradation rate of AFB 1 in the culture filtrate was 85.5%, which showed the feasibility of applying A. niger FS10 culture filtrate to control AFB 1 contamination in fermented pastes.
Saccharomyces cerevisiae is one of the most important yeasts to produce various volatile aroma compounds, including acetate ester, which plays a significant role in the flavor formation of fermented pastes [81]. S. cerevisiae was revealed to be a potential biocontrol agent. For example, both S. cerevisiae EBF101 and S. cerevisiae 117 were proven to inhibit the growth of A. flavus Z103 by up to 85% and 83%, respectively [82]. Moreover, 1,2-benzenedicarboxylic acid dioctyl ester, which is one of the primary metabolites of S. cerevisiae, possesses antifungal and antibacterial properties (Figure 4).

Physicochemical Control Methods
Different grains make up fermented pastes. Various Aspergillus fungi strains generally exist after long-term grain storage. Hull-less crops have higher levels of AFB 1 accumulation than hulled crops during storage [83]. Environmental conditions, such as temperature and water activity (A W ), were confirmed to affect the production of mycotoxins by inhibiting the expression of related genes [84]. Regardless of temperature, AFB 1 could not be generated at an A W of 0.82 [85]. Furthermore, the optimal temperatures of mycotoxin production ranged from 25 to 30 • C [86]. In general, temperature and A W are considered to be the most important factors in the management of crop storage. By controlling the A W to be stable and lower than 0.82 and the temperature to be lower than 37 • C, mycotoxins can be effectively inhibited at the source.
Most fermented pastes in China are made up of soybeans, which need to be soaked for 6-18 h in water. Adding organic acids (1 N citric acid and tartaric acid) during the soaking process reduced AFB 1 by 94.1% and 95.1%, respectively [87]. It is well known that AFB 1 is converted into β-keto acid and then to AFD 1 , which can be hydrolyzed [88]. Organic acids cannot impact the characteristics of various materials. Acidic or alkaline pH levels were shown to be effective in reducing the total amount of aflatoxins. Also the process of autoclaving significantly decreased the level of AFB 1 at different pH levels compared to non-autoclaved samples [87] (Figure 4).

Detection Methods of Mycotoxins
At present, the main technologies regarding the determination of mycotoxins are based on chromatography, such as HPLC [89] and LC-MS/MS [90]. The methods of sample extraction include magnetic solid-phase extraction (MSPE) [91], solid-phase extraction (SPE) [92], and liquid-liquid extraction (LLE) [93]. Immunoassays are widely accepted to be able to detect mycotoxins sensitively in a variety of foods and feeds [94]. However, the techniques mentioned above include complicated and separated processes. Many novel methods exist which are simpler and more accurate.

Novel Magnetic Adsorbent Techniques
MSPE is a widely used method involving magnetic adsorption, whereby a product can be isolated from a sample solution using a magnet after the adsorption of analytes [95]. Recent studies focused on a new carbon-based nanomaterial named graphene as a magnetic adsorbent, which possesses unique properties such as strong adsorption capacity and excellent chemical stability. Furthermore, 3D graphene (3D-G), which has a low-density porous structure and versatile functionalization, overcomes the shortcomings of 2D graphene (2D-G) [96].
A new type of magnetic SPE, named graphene-coated magnetic-sheet SPE, was developed, in which magnetic 3D-G@Fe 3 O 4 nanoparticles fixed on a perforated magnetic sheet were placed in a syringe filter holder and used as the adsorbent phase for the first time [97]. This technique was used to extract AFB 1 , B 2 , G 1 , and G 2 rapidly, i.e., within 30 min, from fermented pastes. The relative recoveries of graphene-coated magnetic-sheet SPE were 83-102%, and the reusability was 20 times without significant loss of extraction recovery (< 4%).
Furthermore, the novel absorbent used an aptamer to functionalize magnetic agarosemicrospheres (MAMs) and bind to AFBs, then immobilized the AFB-aptamers on MAMs via a biotin-streptavidin interaction. The LOD values were 25 pg/mL for AFB 1 and 10 pg/mL for AFB 2 under reasonable conditions [99].

Aptamer Probes Techniques
Aptamers are specific affinity reagents with small sequences of oligonucleotides that can be screened for in vitro [100] and mimic antibodies by folding into complex 3D shapes that bind to specific targets [101]. Several works investigated aptamers in terms of designing them to construct various detection methods, such as the rapid detection of zearalenone in corn samples [102] and chloramphenicol determination [103]. Their best advantage is that they can be designed into numerous structures, including oligonucleotide probes to output signals directly.
An electrochemical aptasensor for aflatoxin B (AFB) detection using cyclic voltammetry (CV) and electochemical impedance spectroscopy (EIS) was developed with high sensitivity and selectivity. This aptasensor can be applied to detect AFB1 in white wine, peanuts, cashew nuts, and soy sauce, with a recovery of 85-100% [104].
In order to amplify the signal of aptamer probes, a novel AFB 1 aptamer probe was studied using complementary DNA as a signal generator for amplification using real-time quantitative polymerase chain reaction (PCR) [105]. Many amplification strategies were adopted, involving complex enzyme catalysis and precise temperature control, which hindered the application of on-the-spot aflatoxin detection. Xia et al. proposed an aptamer probe design strategy to achieve AFB 1 detection with enzyme-free amplification and homogeneous reactions [106], which was applied successfully in broad bean paste and peanut oil within one minute, which is the quickest detection of AFB 1 so far.
Besides detecting AFBs, the aptamer was regarded as a high-affinity, specific method to detect OTA [107]. For example, the detection platform for OTA based on fluorescence aptamers was established, and exonuclease III (Exo III) was used to amplify signals and gold nanoparticles (AuNPs) to reduce fluorescence [108]. This aptasensor was confirmed to have excellent selectivity, with a limit of detection of 4.82 nM. Methods based on homogeneous electrochemical sensing protocol using a thionine-aptamer/graphene nanocomplex were also developed. The electronic signal starts off weak, but after making a connection with OTA, the thionine-aptamer/OTA generates an electrochemical signal using to detect OTA contamination in foods [109]. Moreover, biosensors using core-satellite nanostructures were applied to quantify mycotoxins, whereby the chiral intensity weakened by increasing OTA [110].
In recent years, a highly selective method integrating the electrochemistry and chemiluminescence of aptamers, electrochemiluminescence (ECL), has been widely used. Taking advantage of cadmium sulfide semiconductor quantum dots (CdS QDs) and DNA walkers, a novel technique based on the ECL aptasensor was confirmed to be a highly sensitive piece of equipment to quantify OTA during production, with a detection limit of 0.012 nM [111].

On-Site Test
Immunoassays are conventional techniques used AFB 1 pretreatment in many foods. Due to this process being time-consuming and complex, many novel methods based on immunoassaying were developed. Immunomagnetism for pretreatment and enzyme-linked immunosorbent assay (ELISA) for quantification were established to detect AFB 1 in soybean pastes [112]. These methods combined the active superparamagnetic beads and anti-AFB 1 monoclonal antibodies to form immunomagnetic beads (IMBs). The magnetic beads coupled with the antibodies bind to a target material via an affinity reaction. The average recovery was between 0.5 and 7 µg/kg, or 83.6%~104%, and the relative standard deviation was 4.2%~11.7%.
A rapid on-site test for a wide range of aflatoxins in fermented soybean pastes is necessary. A novel strip test was developed based on lateral flow immunoassay, in which 3B6 was used to detect aflatoxins B 1 , M 1 , G 1 , G 2 , and B 2 , which belonged to monoclonal antibodies. The colloidal gold particles coated with 3B6 were considered to be the detector reagent, whereas the AFB 1 -BSA conjugate was regarded as a competitor reagent. This method was applied as a rapid and cost-effective screening tool, which had a limit of detection (0.5 µg/kg) within 10 min [113].

Molecular-Based Techniques
In recent years, molecular-based techniques such as PCR have been widely applied to detect B. cereus through the amplification of specific cereulide genes [114]. However, post-PCR analysis and gel electrophoresis assays increase the possibility of cross-contamination [115]. Furthermore, quantitative real-time PCR (qPCR), which uses intercalating dyes such as SYTO9 or dual-labeled fluorescence probes, offers simultaneous amplification and target-gene detection, which traditional PCR does not [116]. Many works were based on qPCR. For instance, the multiplex qPCR technique was developed to detect different pathogens simultaneously, including B. cereus in a variety of foods. Based on targeting various specific genes as indicators in a single reaction, the multiplex qPCR assay showed high efficiency and accuracy with a detection limit of 10 3 CFU/g for every pathogen [117].
Nevertheless, recent studies demonstrated the limitations of PCR-based methods, which cannot identify DNA from cells, regardless of viability. DNA amplification in nonviable cells leads to overestimation of bacterial counts [118]. Some researchers used propidium monoazide (PMA) as a nucleic acid to overcome this limitation by combining PMA with qRNA to detect and quantify B. cereus in ultra-high-temperature (UHT) milk samples, where the detection limit was 7.5 × 10 2 CFU/mL. Due to cell apoptosis during the UHT process, the detection percentage of PMA-qPCR was 32.6% compared with 57.8% of traditional qPCR, indicating that the application of PMA treatment prevented the overestimation of B. cereus in food [119]. However, the techniques mentioned above either require dual-labeled probes or a separate probe, which are very expensive. A novel pentaplex RT-PCR high-resolution melt-curve assay using DNA intercalating dye SYTO9 was developed [120], which has the ability to quantify four enterotoxin genes, namely nheA, entFM, cytK, and hblD, simultaneously, as well as ces, thereby allowing a wide range of detection of B. cereus. Furthermore, the detection limit in foods is 10 1 CFU/g with seven hours of enrichment. This investigation revealed an economical alternative method for melt-curve analysis and probe-based real-time detection technology.
Although PCR assays are sensitive enough and widely applied in cereulide detection, the fact that B. cereus strains have widely distributed genes presents a limitation [60]. A recent study demonstrated that expanding the number of genes though whole genome sequencing (WGS) was considered to be a more powerful technique than PCR-based methods [121]. Therefore, using WGS as pre-screening measure and BTyper as a data analysis tool would be a useful method to detect toxin genes in mixed cultures.

Conclusions
Three kinds of mycotoxins, namely, aflatoxins, ochratoxins, and cereulide, are often detected in fermented pastes. Different harvest stages, appropriate drying processes, relatively low temperatures, and organic acid pretreatments are the primary influential factors which will significantly help to guide fermentation industries to effectively avoid mycotoxin contamination. This review provided feasible suggestions for effective control, such as biocontrol agents and rapid detection methods throughout the whole production process of fermented pastes.

Conflicts of Interest:
The authors declare no conflict of interest.