Evaluation of sterols as markers of fungal spoilage in red pepper powder.

Red pepper powder (RPP) made from ground dried red pepper (Capsicum annuum L.) is prone to adulteration with fungal-spoiled RPP to gain unfair profits in Korea. This study aimed to investigate the effects of fungal infection on the ergosterol and phytosterol content of RPP and evaluate the potential of the sterol content as a marker for identifying fungal-spoiled RPP. Ergosterol was detected only in fungal-spoiled RPP and not in unspoiled RPP [

Hepa-ToxMOA: a pathway-screening method for evaluating cellular stress and hepatic metabolic-dependent toxicity of natural products.

In the field of drug discovery, natural products have emerged as therapeutic agents for diseases such as cancer. However, their potential toxicity poses significant obstacles in the developing effective drug candidates. To overcome this limitation, we propose a pathway-screening method based on imaging analysis to evaluate cellular stress caused by natural products. We have established a cellular stress sensing system, named Hepa-ToxMOA, which utilizes HepG2 cells expressing green fluorescent protein (GFP) fluorescence under the control of transcription factor response elements (TREs) for transcription factors (AP1, P53, Nrf2, and NF-κB). Additionally, to augment the drug metabolic activity of the HepG2 cell line, we evaluated the cytotoxicity of 40 natural products with and without S9 fraction-based metabolic activity. Our finding revealed different activities of Hepa-ToxMOA depending on metabolic or non-metabolic activity, highlighting the involvement of specific cellular stress pathways. Our results suggest that developing a Hepa-ToxMOA system based on activity of drug metabolizing enzyme provides crucial insights into the molecular mechanisms initiating cellular stress during liver toxicity screening for natural products. The pathway-screening method addresses challenges related to the potential toxicity of natural products, advancing their translation into viable therapeutic agents.

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.

Occurrence and risk assessment of okadaic acid, dinophysistoxin-1, dinophysistoxin-2, and dinophysistoxin-3 in seafood from South Korea.

The okadaic acid (OA)-group toxins, including OA, dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), and dinophysistoxin-3 (DTX3), cause diarrheic shellfish poisoning in humans. To manage OA-group toxins more strictly, Korean regulations were recently revised to consider OA, DTX1, DTX2, and DTX3 combined. Thus, our study characterized the occurrence of OA, DTX1, DTX2, and DTX3 in seafood distributed across South Korea, and a risk assessment of seafood consumption was conducted. Two hundred and seventeen samples from 16 bivalve and 7 non-bivalve species collected from three representative coastal areas in 2021 were analyzed via liquid chromatography-tandem mass spectrometry. OA, DTX1, and DTX3 were detected in 2.3%, 4.1%, and 9.2% of the examined samples, with positive mean levels of 11.3, 16.4, and 40.9 µg/kg, respectively. DTX2 was not detected in any of the samples. At least one OA-group toxin was detected in the bivalve samples, including blood clams, pan shells, hard clams, mussels, and scallops, whereas none were detected in non-bivalves. The estimated acute exposure to OA-group toxins through the intake of seafood in the Korean population and consumer groups was low, ranging from 24.7 to 74.5% of the recommended acute reference dose (ARfD) of 0.33 µg OA equivalents/kg body weight. However, for the scallop consumers aged 7-12 years, acute exposure to OA-group toxins exceeded the ARfD, indicating a possible health risk. These results suggest that including DTX3 in the new regulatory limits is appropriate to protect Korean seafood consumers from exposure to OA-group toxins.

Okadaic Acid Is at Least as Toxic as Dinophysistoxin-1 after Repeated Administration to Mice by Gavage.

Okadaic acid (OA) and its analogues cause diarrhetic shellfish poisoning (DSP) in humans, and risk assessments of these toxins require toxicity equivalency factors (TEFs), which represent the relative toxicities of analogues. However, no human death by DSP toxin has been reported, and its current TEF value is based on acute lethality. To properly reflect the symptoms of DSP, such as diarrhea without death, the chronic toxicity of DSP toxins at sublethal doses should be considered. In this study, we obtained acute oral LD50 values for OA and dinophysistoxin-1 (DTX-1) (1069 and 897 µg/kg, respectively) to set sublethal doses. Mice were treated with sublethal doses of OA and DTX-1 for 7 days. The mice lost body weight, and the disease activity index and intestinal crypt depths increased. Furthermore, these changes were more severe in OA-treated mice than in the DTX-1-treated mice. Strikingly, ascites was observed, and its severity was greater in mice treated with OA. Our findings suggest that OA is at least as toxic as DTX-1 after repeated oral administration at a low dose. This is the first study to compare repeated oral dosing of DSP toxins. Further sub-chronic and chronic studies are warranted to determine appropriate TEF values for DSP toxins.

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells.

Emodin, an emerging mycotoxin, is known to be hepatotoxic, but its mechanism remains unclear. We hypothesized that emodin could induce endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway and apoptosis, which are closely correlated and contribute to hepatotoxicity. To test this hypothesis, a novel IRE1α inhibitor, STF-083010, was used. An MTT assay was used to evaluate metabolic activity, and quantitative PCR and western blotting were used to investigate the gene and protein expression of ER stress or apoptosis-related markers. Apoptosis was evaluated with flow cytometry. Results showed that emodin induced cytotoxicity in a dose-dependent manner in HepG2 cells and upregulated the expression of binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), IRE1α, spliced XBP1, the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and cleaved caspase-3. Cotreatment with emodin and STF-083010 led to the downregulation of BiP and upregulation of CHOP, the Bax/Bcl-2 ratio, and cleaved caspase-3 compared with single treatment with emodin. Furthermore, the apoptosis rate was increased in a dose-dependent manner with emodin treatment. Thus, emodin induced ER stress in HepG2 cells by activating the IRE1α-XBP1 axis and induced apoptosis, indicating that emodin can cause hepatotoxicity.

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.

Antibody-free and selective detection of okadaic acid using an affinity peptide-based indirect assay.

Okadaic acid (OA) is a type of marine biotoxin produced by some species of dinoflagellates in marine environments. Consumption of shellfish contaminated with OA can cause diarrhetic shellfish poisoning (DSP) in humans with symptoms that typically include abdominal pain, diarrhea and vomiting. In this study, we developed an affinity peptide-based direct competition enzyme-linked immunosorbent assay (dc-ELISA) for the detection of OA in real samples. The OA-specific peptide was successfully identified via M13 biopanning and a series of peptides were chemically synthesized and characterized their recognition activities. The dc-ELISA system showed good sensitivity and selectivity with a half-maximal inhibitory concentration (IC50) of 148.7 ng/mL and a limit of detection (LOD) of 5.41 ng/mL (equivalent, 21.52 ng/g). Moreover, the effectiveness of the developed dc-ELISA was validated using OA-spiked shellfish samples, and the developed dc-ELISA showed a high recovery rate. These results suggest that the affinity peptide-based dc-ELISA can be a promising tool for detecting OA in shellfish samples.

Degradation mechanism of aflatoxin B1 and aflatoxin G1 by salt tolerant Bacillus albus YUN5 isolated from 'doenjang', a traditional Korean food.

Aflatoxins are the mycotoxins that contaminate food and feed and pose health hazards to humans and animals. Here, Bacillus albusYUN5 was isolated from doenjang (Korean fermented soybean paste) and examined for aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) degradation capabilities. The highest degradation of AFB1 (76.28 ± 0.15%) and AFG1 (98.98 ± 0.00%) was observed in the cell-free supernatant (CFS) ofB. albusYUN5, whereas negligible degradation was observed in intracellular fraction, viable cells, and cell debris. Furthermore, heat (100 °C) and proteinase K treated CFS possessed AFB1 and AFG1 degradation ability, suggesting that substances other than proteins or enzymes are responsible for the degradation. Optimal degradation of AFB1 and AFG1 by the CFS was achieved at 55 °C and 45 °C, respectively, and at pH 7-10 and salt concentration of 0-20%. Liquid chromatography-mass spectroscopy analysis of the degraded products revealed that either the difuran or lactone ring of AFB1 and lactone ring of AFG1 is the main target site by CFS of B. albus YUN5. A slightly better reduction of AFB1 and AFG1 was observed in doenjang treated with CFS and viable cells of B. albus YUN5 compared to those without CFS and B. albus YUN5 treated doenjang during one year of fermentation, suggesting the applicability of B. albus in real food.

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.

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.

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.

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.

Resilience of Chemistry: An Introduction to the Agricultural and Food Chemistry Technical Program at the 262nd American Chemical Society National Hybrid Meeting & Exposition, Both Online and in Atlanta, Georgia.

This is the third special issue of the Journal of Agricultural and Food Chemistry (JAFC) based on the Agricultural and Food Chemistry Division (AGFD) technical program, at the 262nd American Chemical Society National Meeting. This was the first national meeting held in a hybrid format, both virtually and in-person in Atlanta, Georgia, U.S.A., on August 22-26, 2021. The AGFD proudly hosted 12 symposia, including three award symposia. There were 34 sessions held in total, with 143 oral presentations and 49 poster presentations. This meeting was highly successful in terms of attendance, and technology issues experienced at the previous virtual meetings were successfully resolved.

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.

Aflatoxin Reduction and Retardation of Aflatoxin Production by Microorganisms in Doenjang during a One-Year Fermentation.

Meju, a raw material for doenjang preparation, is highly vulnerable to aflatoxin-producing fungi. The aim of this study was to evaluate the effect of a one-year fermentation on aflatoxins and aflatoxin-producing fungi in doenjang spiked with aflatoxins B1, G1, B2, and G2 and inoculated with toxigenic Aspergillus flavus. A significant reduction in aflatoxins was observed after a year of fermentation, measuring 92.58%, 100%, 98.69%, and 100% of B1, G1, B2, and G2, respectively. After a year of fermentation, 6.95 ± 3.64 µg/kg of total aflatoxin was detected, which represents a 97.88% reduction in the total aflatoxin compared with the initial value (328.83 ± 36.60 µg/kg). Several aflatoxin-degrading fungi (Aspergillus versicolor, Cladosporium subcinereum, Aspergillus ochraceus) and bacteria (Bacillus albus, Bacillus velezensis) isolated from doenjang were identified as the major contributors to the reduction of aflatoxin. Furthermore, it was observed that most of the aflatoxin contamination in doenjang occurred during the meju stage, and this stage was found to be most susceptible to A. flavus contamination and growth. These findings reveal that native microorganisms mediate aflatoxin clean-up in doenjang during fermentation and support the use of such microorganisms as a starter culture for the preparation of aflatoxin-free doenjang.

Combined analysis of inorganic elements and sugars for the identification of red pepper powder containing undeclared ingredients.

This study sought to identify adulterated red pepper powder (RPP) containing undeclared ingredients, such as salts, glucose, Monascus red pigments, and other plant ingredients (e.g., wheat flour, garlic powder, onion powder) using element and sugar analysis methods. Analytical data were obtained for 66 samples of authentic RPP and 12 samples of adulterated RPP. The variables selected to identify the authenticity of RPP include Na, Cl, K, maltohexaose, and maltoheptaose, which partly or totally originate from RPP, salts, or Monascus red pigments. All the 12 samples of commercial seasoned RPP used as models of adulterated RPP and all the 20 blind samples containing ≥10% (w/w) of commercial seasoned RPP were correctly identified by applying the range of the five variables found for the authentic RPP samples. Our findings suggest that combined analyses of the above five constituents could be used to identify adulterated RPP containing undeclared ingredients. PRACTICAL APPLICATION: Adulteration of high-priced spice products, including red pepper powder (RPP), has increasingly become a public concern worldwide as it endangers consumer health and represents economic fraud. This study provides analytical methods that can accurately determine the authenticity of RPP. They would become effective means for protecting producers and suppliers against unfair competition and consumers against health threats.

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.

Distinguishing Korean and Chinese red pepper powder using inductively coupled plasma and X-ray fluorescence-based analysis.

This study aimed to distinguish between Korean and Chinese red pepper powder (RPP) using inorganic elemental analysis data combined with orthogonal partial least squares-discriminant analysis (OPLS-DA). Elemental concentrations were obtained for 31 Korean and 31 Chinese RPP samples that were collected in Korea. Energy dispersive X-ray fluorescence spectroscopy detected 11 elements in these samples. Rb and Cl concentrations were selected as the variables which best allowed distinguishing between Korean and Chinese RPP using an S-plot from OPLS-DA. Rb and Cl concentrations in the Korean RPP samples were ≤ 1.6 mg/100 g (measured by inductively coupled plasma-optical emission spectroscopy) and ≤ 215 mg/100 g, respectively. A blind trial demonstrated that Korean RPP containing ≥ 50 g/100 g of Chinese RPP could be identified by applying predetermined ranges of Rb and Cl concentrations, suggesting that analysis of these two elements is a possible approach to distinguish between Korean and Chinese RPP.

Complete chloroplast genome of Gypsophila oldhamiana Miq. (Caryophyllales: Caryophyllaceae).

The complete chloroplast genome sequence of Gypsophila oldhamiana Miq., a species of the Caryophyllaceae family, was assembled and analyzed from the de novo assembly of Illumina paired-end sequencing data. The total length of the chloroplast genome of G. oldhamiana was 152,675 bp including a large single-copy (LSC) region of 83,552 bp, a small single-copy (SSC) region of 17,349 bp, and a pair of identical inverted repeat regions (IRs) of 25,887 bp. The genome possessed a total of 130 genes, including 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The phylogenetic analysis of G. oldhamiana with 14 related species discovered the closest taxonomical relationship with Gypsophila vaccaria voucher in the Caryophyllaceae family.