Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure.

Aflatoxins are strong carcinogenic and mutagenic fungal metabolites, and aflatoxin contamination is a critical issue in agriculture and food production. Natural flavonoids can suppress aflatoxin biosynthesis; however, the structure-activity relationship remains unclear. In the present study, a total of 36 structurally related natural flavonoids were tested against the aflatoxigenic Aspergillus flavus, both in-vitro and in-situ (on maize kernels), to investigate their structure-activity relationship and biological activity. Aflatoxin production (IC50 values: 10.85-20.09 µg/mL) and the expression of related genes (aflD, aflK, aflQ, and aflR) were found to be strongly inhibited. Structure-activity relationship studies revealed that the [-OH] or [-O-CH3] groups at position 6 of ring A and position 4' of ring B were closely associated with antifungal and antiaflatoxigenic activities. These findings provide valuable information for the development of clean and safe methods to prevent aflatoxin contamination in food.

Mycotoxins in soybean-based foods fermented with filamentous fungi: Occurrence and preventive strategies.

Fermented soybean products are widely consumed worldwide, and their popularity is increasing. Filamentous fungi, such as Actinomucor, Aspergillus, Monascus, Mucor, Penicillium, Rhizopus, and Zymomonas, play critical roles in the fermentation processes of many soybean foods. However, besides producing essential enzymes for food fermentation, filamentous fungi can release undesirable or even toxic metabolites into the food. Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi and may be detected during the food production process. Without effective prevention strategies, mycotoxin contamination in fermented soybean products poses a risk to human health. This review focused on the changes in mycotoxigenic fungal abundance and mycotoxin contamination at different stages during the production of soybean-based fermented foods, as well as effective strategies for preventing mycotoxin contamination in such products. Data from relevant studies demonstrated a tendency of change in the genera of mycotoxigenic fungi and types of mycotoxins (aflatoxins, alternariol, alternariol monomethyl ether, deoxynivalenol, fumonisins, ochratoxin A, rhizoxins, T-2 toxin, and zearalenone) present in the raw materials and the middle and final products. The applicability of traditional chemical and physical mitigation strategies and novel eco-friendly biocontrol approaches to prevent mycotoxin contamination in soybean-based fermented foods were discussed. The present review highlights the risks of mycotoxin contamination during the production of fermented soybean products and recommends promising strategies for eliminating mycotoxin contamination risk in soybean-based fermented foods.

A float culture method for fungal secondary metabolism study using hydrophilic polyvinylidene fluoride membranes.

Fungal metabolism is affected by both the developmental stage and cultivation conditions. Fungal growth in solid culture reflects natural conditions more closely than growth in liquid culture; however, because the mycelium cannot be harvested easily and the medium composition cannot be modified during incubation, the approach has some limitations when compared to liquid culture methods. The float culture incubation method introduced herein enables fungus to develop similar colonies to those on solid culture. This is a simple method that leads to the production of high-quality RNA samples.

Simultaneous determination of 17 regulated and non-regulated Fusarium mycotoxins co-occurring in foodstuffs by UPLC-MS/MS with solid-phase extraction.

Fusarium species produce numerous mycotoxins known to co-occur in food. While some of these mycotoxins (e.g., deoxynivalenol, fumonisins) are regulated in several countries, others are non-regulated (e.g., nivalenol, beauvericin). In this study, UPLC-MS/MS with solid-phase extraction cleanup was used to determine 17 Fusarium mycotoxins (FTs) simultaneously. The method showed excellent performance in terms of linearity (R2 > 0.99), LOD (<1.2 µg/kg), LOQ (<3.6 µg/kg), accuracy (70.0-116.3 %), repeatability (<15.7 %), reproducibility (<25.3 %), and expanded uncertainty (<41.7 %). The validated method was successfully applied to 198 marketed food samples collected in South Korea. Of the tested samples, 79 % were contaminated with at least one FT. Job's tears showed the highest prevalence of 14 FTs, and sorghum had the highest total FTs level (3.03 mg/kg). The results suggest that this method can be used for the simultaneous analysis of 17 FTs in food samples, which would serve as crucial information for risk management.

Risk Assessment Considering the Bioavailability of 3-ß-d-Glucosides of Deoxynivalenol and Nivalenol through Food Intake in Korea.

Deoxynivalenol and nivalenol are major type B trichothecenes and the most frequently occurring mycotoxins worldwide. Their 3-ß-d-glucoside forms have recently become a safety management issue. These glucoside conjugates are converted back to the parent toxins during human digestion, but studies to confirm their bioavailability are lacking. In this study, a risk assessment was performed considering the bioavailability of glucoside conjugates. A literature review was conducted to compile the existing bioavailability studies of glucoside conjugates, and three exposure scenarios considering bioavailability were established. As a result of a risk assessment using deterministic and probabilistic methods, both the deoxynivalenol and nivalenol groups had safe levels of tolerable daily intake percentage (TDI%), not exceeding 100%. The TDI% for the nivalenol group was approximately 2-3 times higher than that for the deoxynivalenol group. Notably, infants showed higher TDI% than adults for both toxin groups. By food processing type, the overall TDI% was highest for raw material, followed by simple-processed and then fermented-processed. Since glucoside conjugates can be converted into parent toxins during the digestion process, a risk assessment considering bioavailability allows the more accurate evaluation of the risk level of glucoside conjugates and can direct their safety management in the future.

Antifungal Activity of Essential Oil and Plant-Derived Natural Compounds against Aspergillus flavus.

Aspergillus flavus is a facultative parasite that contaminates several important food crops at both the pre- and post-harvest stages. Moreover, it is an opportunistic animal and human pathogen that causes aspergillosis diseases. A. flavus also produces the polyketide-derived carcinogenic and mutagenic secondary metabolite aflatoxin, which negatively impacts global food security and threatens human and livestock health. Recently, plant-derived natural compounds and essential oils (EOs) have shown great potential in combatting A. flavus spoilage and aflatoxin contamination. In this review, the in situ antifungal and antiaflatoxigenic properties of EOs are discussed. The mechanisms through which EOs affect A. flavus growth and aflatoxin biosynthesis are then reviewed. Indeed, several involve physical, chemical, or biochemical changes to the cell wall, cell membrane, mitochondria, and related metabolic enzymes and genes. Finally, the future perspectives towards the application of plant-derived natural compounds and EOs in food protection and novel antifungal agent development are discussed. The present review highlights the great potential of plant-derived natural compounds and EOs to protect agricultural commodities and food items from A. flavus spoilage and aflatoxin contamination, along with reducing the threat of aspergillosis diseases.

Simple chromatographic determination of aflatoxins in Korean fermented soybean products doenjang, ganjang, and gochujang, with comparison of derivatization methods.

Korean fermented soybean products doenjang, ganjang, and gochujang are vulnerable to contamination with aflatoxigenic fungi in the open fermentation environment. Therefore, simple and effective methods to determine aflatoxins (AFs) in these complex food matrices are needed. High-performance liquid chromatography with fluorescence detection using two derivatization methods for AF determination in three fermented soybean products was optimized and validated. Pre-column derivatization (preCD) of the AF extracts was performed using trifluoroacetic acid, and post-column derivatization (PCD) was performed in a photochemical reactor for enhanced detection. Both derivatization methods resulted in acceptable performances for linearity (R2 > 0.999), recovery (71-118%), and precision (< 10.6%) values. Recovery and precision with preCD and PCD were similar, but the limit of detection was superior with PCD. When these analytical methods were applied to commercially available fermented soybean products, the AF levels of all commercial products were ranged from not detected to 6.06 µg/kg.

Contamination characteristics and risk assessment of aflatoxins in homemade soybean paste, a traditional fermented soybean food, in South Korea.

Soybean paste is manufactured through microbial fermentation and may become contaminated with aflatoxins. Herein, we conducted nationwide large-scale monitoring (n = 1436) over three years (2018-2020) to investigate aflatoxin levels according to geographic, demographic, manufacturing, quality factors, and risk characteristics of homemade soybean paste produced through fermentation. The mean level of total aflatoxins was 5.88 µg/kg (range, 0.01-281.92), with the most common contaminating type being the B type. Aflatoxin levels significantly differed according to the region, age of the manufacturer, type of starter used, and the amino-type nitrogen content and pH of the homemade soybean paste (p < 0.05). Aflatoxin levels was significantly higher when starters were manufactured using the traditional method (inoculation with a naturally occurring strain in the surrounding environment). The aflatoxin exposure level estimated through the average intake of homemade soybean paste in all age groups was 0.1012 ng/kg body weight/day. The risk assessment for the genotoxic and carcinogenic potential of aflatoxins using the margin of exposure approach revealed values of 3705-3954 for average intake of homemade soybean paste, indicating public health concern. These results suggest that follow-up studies and safety management strategies are needed to reduce aflatoxin levels in homemade soybean paste.

Natural Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods Marketed in South Korea by LC-MS/MS.

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity (R2 > 0.999), the limit of detection (0.04-1.67 µg/kg), the limit of quantification (0.12-5.06 µg/kg), recovery (80.0-114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 µg/kg for AOH, 4.36 µg/kg for AME, 0.14 µg/kg for ALT, 0.11 µg/kg for ATX-I, 0.43 µg/kg for TEN, and 104.56 µg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.

Evaluating the impact of interbasin water transfer on water quality in the recipient river basin with SWAT.

During the second half of the 20th century in South Korea, interbasin water transfers (IBWTs) have been used to supply the water demands of basins with insufficient water using water from reservoirs in neighboring basins with ample water. However, since 2000, frequent droughts have resulted in water resource imbalances in donor basins, and basin residents have begun to claim their water rights. Recipient basins have also experienced water shortages and water quality deterioration due to gradual urban growth, agricultural activities, and climate change impacts. In this study, the Mangyeong River basin (1602 km2) was investigated. This basin has received 380 million m3/year of water since 2002 from the Yongdam multipurpose dam (YDD), which is located in another basin. For IBWT modeling, the Soil and Water Assessment Tool (SWAT) model and an inlet function were applied to model the recipient and donor water quality. Eight scenarios related to water transfer quantity and quality were applied with SWAT to analyze the effects of IBWT on the water quality in the recipient basin. The results showed that an increase in the IBWT amount helped to reduce the nutrient and suspended solids concentrations in the recipient basin when the donor's nutrient and suspended solids concentrations were lower than those in the recipient basin. The IBWT quantity scenarios had a greater impact on the water quality of the recipient basin than the IBWT quality scenarios. These results could provide basic information for use in deciding on the quantity and quality of IBWT between basins that are in conflict.

Transcriptomic responses of Aspergillus flavus to temperature and oxidative stresses during aflatoxin production.

Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.

Effect of plant-based compounds on the antifungal and antiaflatoxigenic efficiency of strobilurins against Aspergillus flavus.

Aflatoxins are a group of carcinogenic and mutagenic fungal secondary metabolites that have threatened human health and global food security. Aflatoxin contamination can be controlled by applying fungicides, such as strobilurins. Although these compounds have been effective, they may be risky to the environment due to their wide usage. In this study, plant-based compounds were tested to promote the performance of strobilurins (azoxystrobin, pyraclostrobin) against aflatoxigenic Aspergillus flavus; six natural compounds, namely baicalein, nobiletin, meso-dihydroguaiaretic acid, pinoresinol, syringaresinol, and celastrol, were found to exhibit synergistic antifungal effects with strobilurins with fractional inhibitory concentration index < 0.5. Among them, baicalein showed no inhibitory effects on A. flavus when applied alone, but strongly enhanced the in vitro and in situ antifungal and antiaflatoxigenic efficacy of strobilurins and transformed them from fungistatic to fungicidal agents. Therefore, baicalein may be used as an effective natural chemosensitizing agent to improve the performance of strobilurins against A. flavus. The findings of this study provide novel insights for the development of safer and more effective strategies for the control of aflatoxin contamination.

Alternative Oxidase: A Potential Target for Controlling Aflatoxin Contamination and Propagation of Aspergillus flavus.

Aflatoxins are among the most hazardous natural cereal contaminants. These mycotoxins are produced by Aspergillus spp. as polyketide secondary metabolites. Aflatoxigenic fungi including A. flavus express the alternative oxidase (AOX), which introduces a branch in the cytochrome-based electron transfer chain by coupling ubiquinol oxidation directly with the reduction of O2 to H2O. AOX is closely associated with fungal pathogenesis, morphogenesis, stress signaling, and drug resistance and, as recently reported, affects the production of mycotoxins such as sterigmatocystin, the penultimate intermediate in aflatoxin B1 biosynthesis. Thus, AOX might be considered a target for controlling the propagation of and aflatoxin contamination by A. flavus. Hence, this review summarizes the current understanding of fungal AOX and the alternative respiration pathway and the development and potential applications of AOX inhibitors. This review indicates that AOX inhibitors, either alone or in combination with current antifungal agents, are potentially applicable for developing novel, effective antifungal strategies. However, considering the conservation of AOX in fungal and plant cells, a deeper understanding of fungal alternative respiration and fungal AOX structure is needed, along with effective fungal-specific AOX inhibitors.

Occurrence of Deoxynivalenol, Nivalenol, and Their Glucosides in Korean Market Foods and Estimation of Their Population Exposure through Food Consumption.

Major type B trichothecene mycotoxins, including deoxynivalenol (DON), nivalenol (NIV), and their respective glucoside conjugates, deoxynivalenol-3-ß-D-glucose (DON3G) and nivalenol-3-ß-D-glucose (NIV3G), are present in food products, such as cereals, legumes, and their processed products. Thus, here, DON, NIV, and their 3-ß-D-glucosides were monitored in 506 Korean market foods, and exposure to these mycotoxins was estimated in the population consuming these foods. The accuracy and precision of our method, which simultaneously determined four toxins, were 80.1-106.5% and 0.3-12.4%, in four representative food matrices assessed. The incidences of DON, DON3G, NIV, and NIV3G among all food samples tested were 13%, 8%, 12%, and 5%, respectively. The glucoside conjugate with free toxin was found to have the maximum co-occurrence of 49%. The estimated daily intakes of DON, DON3G, NIV, and NIV3G through food intake under four different scenarios were 0.019-0.102, 0.004-0.089, 0.007-0.094, and 0.002-0.095 µg kg-1 body weight (b.w.) day-1, respectively, which are lower than the established health-based guidance values. Overall, our results suggest that the estimated exposure of the Korean population to type B trichothecenes, namely, DON, NIV, and their 3-ß-D-glucoside conjugates, may not pose a potential health risk.

Simple validated method for simultaneous determination of deoxynivalenol, nivalenol, and their 3-ß-D-glucosides in baby formula and Korean rice wine via HPLC-UV with immunoaffinity cleanup.

A simple and reliable method for the simultaneous determination of major type B trichothecene mycotoxins, deoxynivalenol (DON) and nivalenol (NIV), along with their 3-ß-d-glucosides (DON-3-glucoside (DON3G) and NIV-3-glucoside (NIV3G)) in baby formula and Korean rice wine was validated in the present study. The method was based on immunoaffinity cleanup followed by analysis using an HPLC-UV technique. The method was validated in-house for two matrices as follows: linearity (R2 > 0.99) was established in the range of 20-1000 µg kg-1; accuracy (expressed as recovery) ranged from 78.7 to 106.5% for all the analytes; good intermediate precision (relative standard deviation < 12%), and adequate detection and quantitation limits (< 4.4 and < 13.3 µg kg-1, respectively) were achieved. Furthermore, the estimated measurement expanded uncertainty was determined to be 4-24%. The validated method was successfully applied to the analysis of 31 baby formulas and Korean rice wines marketed in Korea.

Simultaneous Determination of Twenty Mycotoxins in the Korean Soybean Paste Doenjang by LC-MS/MS with Immunoaffinity Cleanup.

Doenjang, a Korean fermented soybean paste, is vulnerable to contamination by mycotoxins because it is directly exposed to environmental microbiota during fermentation. A method that simultaneously determines 20 mycotoxins in doenjang, including aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), and fumonisins (FBs) with an immunoaffinity column cleanup was optimized and validated in doenjang using LC-MS/MS. The method showed good performance in the analysis of 20 mycotoxins in doenjang with good linearity (R2 > 0.999), intra- and inter-day precision (<16%), recovery (72-112%), matrix effect (87-104%), and measurement uncertainty (<42%). The validated method was applied to investigate mycotoxin contamination levels in commercial and homemade doenjang. The mycotoxins that frequently contaminated doenjang were AFs, OTA, ZEN, and FBs and the average contamination level and number of co-occurring mycotoxins in homemade doenjang were higher than those in commercially produced doenjang.