Fermented traditional Chinese medicine(TCM) has a long history of medicinal use, such as Sojae Semen Praeparatum, Arisaema Cum Bile, Pinelliae Rhizoma Fermentata, red yeast rice, and Jianqu. Fermentation technology was recorded in the earliest TCM work, Shen Nong's Classic of the Materia Medica. Microorganisms are essential components of the fermentation process. However, the contamination of fermented TCM by toxigenic fungi and mycotoxins due to unstandardized fermentation processes seriously affects the quality of TCM and poses a threat to the life and health of consumers. In this paper, the characteristics, microbial composition, and mycotoxin profile of fermented TCM are systematically summarized to provide a theoretical basis for its quality and safety control.
Fermentation ; Mycotoxins/analysis* ; Drugs, Chinese Herbal/analysis* ; Fungi/classification* ; Bacteria/genetics* ; Drug Contamination ; Medicine, Chinese Traditional

BACKGROUND: Among the more than 300 mycotoxins that are known to have toxic effects on animals and humans, Fusarium toxins deoxynivalenol (DON), T-2 and HT-2 toxins (T2/HT2), and zearalenone (ZEN) are frequently detected in domestic agricultural products. This study aimed to assess DON, T2/HT2, and ZEN exposure in Japanese adults by measuring urinary mycotoxins, observing their distributions, and making comparisons with data from other countries. METHODS: A total of 201 individuals participated in the study. Twenty-four-hour urine samples were collected from young adults (34 men and 35 women) in the Tokai region (urban area) and spot urine samples were collected from middle-aged and elderly adults (64 men and 68 women) in the Donan area of Hokkaido Prefecture (rural area). Urinary DON, T2/HT2, and ZEN levels were measured using a validated liquid chromatography-tandem mass spectrometry method. RESULTS: For DON, T2/HT2, and ZEN, the detection frequencies above the limit of detection (LOD) level (0.15, 0.13, and 0.01 µg/L, respectively) in all the samples were 53%, 26%, and 71%, respectively. The median concentrations (95^th percentile) of urinary DON, HT2, and ZEN were 0.19 (3.93), mycotoxins was detected in the urine of 86% of study participants, the concentrations were similar to or lower than those found in other countries (from 19 reports within the past decade). Moreover, the probable daily intake (PDI) values in the present study were lower than the provisional maximum tolerable daily intake levels. The urinary mycotoxin levels did not significantly differ with respect to sex, age, or occupation. CONCLUSIONS This study represents the first comprehensive exposure assessment for DON, T2/HT2, and ZEN in Japanese adults using human biomonitoring methods. These data provide valuable information for a better understanding of mycotoxin exposure in Japan.
Humans ; Zearalenone/urine* ; Japan ; Male ; Female ; Trichothecenes/urine* ; T-2 Toxin/urine* ; Biological Monitoring ; Adult ; Middle Aged ; Aged ; Cross-Sectional Studies ; Young Adult ; Mycotoxins/urine* ; Environmental Exposure/analysis* ; East Asian People

An analytical method for 10 mycotoxins in Hippophae Fructus medicinal and edible products was established in this study, and the contamination of their mycotoxins was analyzed. First of all, the mixed reference solution of ten mycotoxins such as aflatoxin, ochratoxin, zearalenone, and dexoynivalenol was selected as the control, and the Hippophae Fructus medicinal and edible products were prepared. Secondly, based on the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) technology, 10 mycotoxins in Hippophae Fructus medicinal and edible products were quantitatively investigated and their content was determined. Finally, the contamination of mycotoxins was analyzed and evaluated. The optimal analysis conditions were determined, and the methodological inspection results showed that the 10 mycotoxins established a good linear relationship(r>0.99). The method had good repeatability, test sample specificity, stability, and instrument precision. The average recovery rates of 10 mycotoxins in Hippophae Fructus medicinal products, edible solids, and edible liquids were 90.31%-109.4%, 87.86%-107.8%, and 85.61%-109.1%, respectively. Relative standard deviation(RSD) values were 0.22%-10%, 0.75%-13%, and 0.84%-8.5%, repsectively. Based on UPLC-MS/MS technology, the simultaneous determination method for the limits of 10 mycotoxins established in this study has fast detection speed, less matrix interference, high sensitivity, and accurate results, which is suitable for the limit examination of 10 mycoto-xins in Hippophae Fructus medicinal and edible products.
Mycotoxins/analysis* ; Chromatography, Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Hippophae ; Limit of Detection ; Chromatography, High Pressure Liquid/methods*

By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 μg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.
Humans ; Mycotoxins/analysis* ; Coix ; Aflatoxin B1/analysis* ; Chromatography, Liquid/methods* ; Food Contamination/analysis* ; Tandem Mass Spectrometry/methods*

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 μm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 μg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 μg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.
Aflatoxins/analysis* ; Chromatography, High Pressure Liquid/methods* ; Drug Contamination ; Food Contamination/analysis* ; Mycotoxins/analysis* ; Plant Roots/chemistry* ; Tandem Mass Spectrometry/methods*

In the process of harvesting, production and processing, storage, and transportation, the traditional Chinese medicine Platycladi Semen is prone to mildew due to its own and environmental factors, which can nourish the production of toxic or pathogenic fungi, and even produce mycotoxins, which affects the safety of clinical medication. The 2020 edition of Chinese Pharmacopoeia limits the highest standard of aflatoxin content in Platycladi Semen. However, there are few studies on the fungal contamination of Platycladi Semen, and it is difficult to prevent and control it in a targeted manner. Therefore, based on the Illumina NovaSeq6000 platform, this article uses ITS sequence amplicon technology to analyze the distribution and diversity of fungi in 27 batches of commercially available Platycladi Semen in the Chinese market. A total of 10 phyla, 35 classes, 93 orders, 193 families, 336 genera, and 372 species of fungi were identified in China. Among them, Aspergillus, Alternaria spp. were dominant, 20 batches of samples were detected for A. flavus, 10 batches of samples were detected for A. nidulans, and all samples were detected for potential pathogenic fungi such as A. fumigatus and A. niger. According to diversity analysis, the diversity of the fungal communities in the samples from Gansu province was high, the samples in Shandong province contain the largest number of fungal species, and the samples in Guangxi province had the lo-west diversity and the least number of species. In most samples, pathogenic fungi such as A. fumigatus, A. niger, A. flavus, A. parasiticus were detected in varying degrees. This study systematically investigated the fungal contamination of Platycladi Semen from the markets in the last link of the its industrial chain, and clarified the distribution of Platycladi Semen fungi, especially toxin-producing fungi, and provided theoretical basis for the targeted prevention and control of fungal contamination in Platycladi Semen.
Aflatoxins ; China ; Fungi/genetics* ; Humans ; Mycobiome ; Mycotoxins/analysis* ; Semen/chemistry*

A suitable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of 11 mycotoxins with isotope internal standard in malt. The mycotoxins in malt were extracted and purified by one-step ultrasonic extraction procedure using acetonitrile/water/acetic acid (80 : 19 : 1), and then detected and confirmed by UPLC-MS/MS, and quantified by isotope labeled AFB1 ([13C17]-AFB1) and ZEN ([13C18]-ZEN) internal standards. Rapid separation of the 11 mycotoxins was successfully achieved on a Phenomenex Kinetex C18 column (100 mm x 2.1 mm, 2.6 μm) with gradient elution using the mobile phase of methanol containing 0.1% formic acid and 2 mmol x L(-1) ammonium acetate in water. Simultaneous acquisition was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) source operated in both positive and negative ionization modes. The established method provided a good linearity for the 11 mycotoxins within their respective linear ranges with correlation coefficients all higher than 0.999 1. The average recoveries ranged from 75.0% to 117.0% with relative standard deviations (RSDs) below 5.1%. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.05 to 30 μg x kg(-1) and 0.15 to 87.5 μg x kg(-1), respectively, which were below the maximum residue levels (MRLs) set by the European Union. Twenty malt samples were analyzed and nine samples were detected with mycotoxins, which were confirmed according to the same fragment ions found in positive samples and the standards at the same retention time. This study has demonstrated that the one-step extraction procedure of mycotoxins from complex matrices coupled to UPLC-MS/MS method is simple, quick, accurate and sensitive for quantitative and qualitative analysis of multiple mycotoxins in malt.
Chromatography, High Pressure Liquid ; Fermentation ; Hordeum ; chemistry ; Limit of Detection ; Mycotoxins ; analysis ; isolation & purification ; Tandem Mass Spectrometry

To ensure the quality and safety of Panax notoginseng, a method for the simultaneous determination of 10 mycotoxins in Panax notoginseng was developed using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The sample was extracted with acetonitrile and purified by HLB multifunction cleanup column. The separation was performed on a Phenomenex Kinetex XB-C18 column by gradient elution using methanol and 5 mmol·L(-1) ammonium acetate as mobile phase. The targeted compounds were detected in MRM mode by mass spectrometry with electrospray ionization (ESI) source operated in both positive and negative ionization modes. The linear relationships of the 10 mycotoxins were good in their respective linear ranges. The correlation coefficients (r) ranged from 0.9981 to 1.0000. The LOQs of the 10 mycotoxins were between 0.15 and 8.6 μg·kg(-1). The average recoveries ranged from 73.8% to 107.0% with relative standard deviations (RSDs) of 0.10%-10.9%. The results demonstrated that the proposed method was sensitive and accurate, and suitable for the mycotoxins quantification in Panax notoginseng.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Drug Contamination ; Mycotoxins ; analysis ; Panax notoginseng ; chemistry ; Tandem Mass Spectrometry

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.
Aflatoxin B1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Fluorescent Dyes ; chemistry ; Fumonisins ; analysis ; Mycotoxins ; analysis ; Ochratoxins ; Organic Chemicals ; chemistry ; Staining and Labeling ; Zea mays

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.
Animal Feed/analysis ; Animals ; Antibodies, Fungal/analysis ; Antibodies, Monoclonal/analysis ; Chemistry Techniques, Analytical/*methods ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Female ; Food Contamination/*analysis ; Fusarium/immunology ; Imidazoles/chemistry ; Magnetics/methods ; Mice ; Mice, Inbred BALB C ; Mycotoxins/*analysis/chemistry ; Nanoparticles/chemistry ; Ovalbumin/chemistry ; Trichothecenes/*analysis/chemistry