Occurrence of fungi and mycotoxins in herbal medicines and rapid detection of toxin-producing fungi.

Contamination from external hazardous materials may greatly influence the safety and efficacy of herbal medicines. This paper aimed to evaluate the levels of contamination by mycotoxins and toxigenic fungi in herbal medicines and establish a rapid method for detecting toxin-producing fungi. Herein, 62.92%, 36.25%, and 64.17% of herbal medicines were contaminated by aflatoxins (AFs), ochratoxins, and fumonisins, respectively. Aspergillus (43.77%), Fusarium (5.17%), and Cladosporium (4.46%) were the three predominant genera. Spearman's correlation results showed that Aspergillus and Fusarium were significantly and positively correlated with mycotoxin content (R > 0.5, P < 0.05). In addition, 323 fungal strains were isolated from herbal medicines, and 20 species were identified, mainly belonging to Aspergillus and Penicillium. Analysis of potential mycotoxin-producing fungi showed that Aspergillus flavus can produce AFs, and Aspergillus ochraceus and Aspergillus niger can produce ochratoxin A (OTA). Multiplex real-time polymerase chain reaction showed that A. flavus harbored AF synthesis genes (aflR), and A. ochraceus and A. niger harbored OTA synthesis genes (aoksl). With these synthesis genes, 67.07% and 37.20% of 164 herbal medicines were positive for toxigenic genes. Furthermore, an excellent correlation was found between the above gene copies and mycotoxin content (R2 = 0.99). Our results confirmed the high detection rate of mycotoxins in herbal medicines and identified pivotal AF- and OTA-producing fungi. In conclusion, this paper provided the contamination status of fungi and mycotoxins in herbal medicines and established a rapid method for detecting toxigenic fungi.

Multiple Mycotoxin Contamination in Medicinal Plants Frequently Sold in the Free State Province, South Africa Detected Using UPLC-ESI-MS/MS.

Medicinal plants are important in the South African traditional healthcare system, the growth in the consumption has led to increase in trade through muthi shops and street vendors. Medicinal plants are prone to contamination with fungi and their mycotoxins. The study investigated multiple mycotoxin contamination using Ultra High Pressure Liquid Chromatography-Tandem Mass Spectrometry (UPLC-ESI-MS/MS) for the simultaneous detection of Aflatoxin B1 (AFB1), Deoxynivalenol (DON), Fumonisins (FB1, FB2, FB3), Nivalenol (NIV), Ochratoxin A (OTA) and Zearalenone (ZEN) in frequently sold medicinal plants. Medicinal plant samples (n = 34) were purchased and analyzed for the presence of eight mycotoxins. DON and NIV were not detected in all samples analyzed. Ten out of thirty-four samples tested positive for mycotoxins -AFB1 (10.0%); OTA (10.0%); FB1 (30.0%); FB2 (50.0%); FB3 (20.0%); and ZEN (30.0%). Mean concentration levels ranged from AFB1 (15 µg/kg), OTA (4 µg/kg), FB1 (7-12 µg/kg), FB2 (1-18 µg/kg), FB3 (1-15 µg/kg) and ZEN (7-183 µg/kg). Multiple mycotoxin contamination was observed in 30% of the positive samples with fumonisins. The concentration of AFB1 reported in this study is above the permissible limit for AFB1 (5 µg/kg). Fumonisin concentration did not exceed the limits set for raw maize grain (4000 µg/kg of FB1 and FB2). ZEN and OTA are not regulated in South Africa. The findings indicate the prevalence of mycotoxin contamination in frequently traded medicinal plants that poses a health risk to consumers. There is therefore a need for routine monitoring of multiple mycotoxin contamination, human exposure assessments using biomarker analysis and establishment of regulations and standards.

Preparation of 3-O-, 5-O- and N-palmitoyl derivatives of fumonisin B1 toxin and their characterisation with NMR and LC-HRMS methods.

We have previously published six esterified O-acyl (EFB1) and three N-acyl fumonisin B1 derivatives extracted from rice cultures inoculated with Fusarium verticillioides, amongst these the identification of N-palmitoyl-FB1 has been clearly established in a spiking experiment. At that time, it was assumed that as in the case of O-acyl-FB1 derivatives, linoleic-, oleic- or palmitic acid esterify through the OH group on the 3C or 5C atom of the carbon chain of the fumonisins. In our most recent experiments, we have synthetically acylated the FB1 toxin and subsequently purified 3-O-palmitoyl- and 5-O-palmitoyl-FB1 toxins in addition to the N-palmitoyl-FB1 toxin. They were identified and characterised using 1H and 13C NMR as well as LC-HRMS. Our aim was the identification of the previously detected O-acyl-FB1 derivatives over the course of a spiking experiment, which were obtained through the solid-phase fermentation of Fusarium verticillioides. By spiking the three synthesized and identified components one-by-one into the fungal culture extract and analysing these cultures using LC-MS, it was clearly demonstrated that the F. verticillioides strain produced both the 5-O-palmitoyl-FB1 and N-palmitoyl-FB1 toxins, but did not produce 3-O-palmitoyl-FB1. Thus, it is highly probable that the components thought to be 3-O-acyl-(linoleoyl-, oleoyl-, palmitoyl-) FB1 derivatives in our previous communication are presumably 10-O-acyl-FB1 derivatives. Since these acylated FB1 derivatives can occur naturally in e.g. maize, the use of these synthesized components as reference materials is of great importance in order to obtain accurate qualitative and quantitative data on the occurrence of acylated fumonisins in different matrices including maize based feed samples. The production of these substances has also made it possible to test their toxicity in cell culture and small animal experiments.

Establishment of an Improved ELONA Method for Detecting Fumonisin B1 Based on Aptamers and Hemin-CDs Conjugates.

Fumonisin B1 (FB1) is a strong mycotoxin that is ubiquitous in agricultural products. The establishment of rapid detection methods is an important means to prevent and control FB1 contamination. In this study, an improved enzyme-linked oligonucleotide assay (ELONA) method was designed and tested to detect the contents of FB1 in maize (corn) samples. F10 modified with biotin was bound to an enzyme label plate that was coated with streptavidin (SA) in advance, and carbon dots (CDs) were used to catalyze the color of tetramethylbenzidine (TMB). The complementary chain of F10 was modified with an amino group and coupled with CDs to obtain conjugates. The sample and conjugates were then added to the enzyme plate coated with F10 (an FB1 aptamer). Upon completion of the color reaction, the absorbance was measured at 450 nm. The LOD of this method was 4.30 ng/mL and the LOQ was 13.03 ng/mL. We observed a linear relationship in the FB1 concentration range of 0-100 ng/mL. The standard curve was y = -0.001482 × x + 0.3463, R2 = 0.9918, and the experimental results could be directly measured visually. The recovery of the maize sample was 97.5-99.23% and 94.54-99.25%, and the total detection time was 1 h.

Mycotoxin Occurrence, Toxicity, and Detoxifying Agents in Pig Production with an Emphasis on Deoxynivalenol.

This review aimed to investigate the occurrence of mycotoxins, their toxic effects, and the detoxifying agents discussed in scientific publications that are related to pig production. Mycotoxins that are of major interest are aflatoxins and Fusarium toxins, such as deoxynivalenol and fumonisins, because of their elevated frequency at a global scale and high occurrence in corn, which is the main feedstuff in pig diets. The toxic effects of aflatoxins, deoxynivalenol, and fumonisins include immune modulation, disruption of intestinal barrier function, and cytotoxicity leading to cell death, which all result in impaired pig performance. Feed additives, such as mycotoxin-detoxifying agents, that are currently available often combine organic and inorganic sources to enhance their adsorbability, immune stimulation, or ability to render mycotoxins less toxic. In summary, mycotoxins present challenges to pig production globally because of their increasing occurrences in recent years and their toxic effects impairing the health and growth of pigs. Effective mycotoxin-detoxifying agents must be used to boost pig health and performance and to improve the sustainable use of crops.

The Role of Fumonisins in the Biological Interaction between Fusarium verticillioides and Sitophilus zeamais.

The aim of the current study was to investigate the entomopathogenic capacity of the mold Fusarium verticillioides and the effect of its mycotoxins fumonisins, on the grain beetle Sitophilus zeamais. We evaluated the capacity of this fungus to infect live insects, the antifungal activity of constituents of the insect's epicuticle, and the effect of a fumonisin extract on the fitness of the insects. We found that F. verticillioides could not penetrate the cuticle of S. zeamais and that the fumonisin extract had no negative effects on the fitness of the insects. However, the progeny of the insects increased, and the fumonisin extract had repellent effects. This is the first report about the effects of fumonisins on the relationship between F. verticillioides and S. zeamais, which may provide useful information about interactions between pathogenic microorganisms and insects, especially on stored product pests.

Flower-like gold nanoparticles-based immunochromatographic test strip for rapid simultaneous detection of fumonisin B1 and deoxynivalenol in Chinese traditional medicine.

A flower-like gold nanoparticles (FGN)-based immunochromatographic test strip (ICS) was developed and used for the first time for the rapid simultaneous detection of fumonisin B1 (FB1) and deoxynivalenol (DON) in Chinese traditional medicine. Several experimental conditions affecting the sensitivity of ICS have been investigated, including the type of FGN, the preparation conditions of FGN-monoclonal antibody (MAb) conjugates, and the process parameters of ICS. Under the optimal experimental conditions, the visual limit of detection was 5.0 ng/mL (corresponding to 50 µg/kg in Chinese traditional medicine samples) for both FB1 and DON, and detection can be completed within 5 min. In addition, the natural samples were analyzed using high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), and the results of these methods showed good correlation with those obtained using ICS. The procedure using FGN-based simultaneous ICS was sensitive, rapid, and convenient for on-site detection of a large number of samples.

A holistic study to understand the detoxification of mycotoxins in maize and impact on its molecular integrity using cold atmospheric plasma treatment.

The need for safe and quality food, free from the presence of hazardous contaminants such as mycotoxins is an on-going and complex challenge. Cold atmospheric pressure plasma (CAPP) has the potential to contribute to achieving this goal. Decontamination efficacy of CAPP against six of the most common mycotoxins found in foods and feedstuffs was assessed herein. Concentration reduction of up to 66% was achieved in maize for both aflatoxin B1 and fumonisin B1. Degradation products were detected only in the case of aflatoxin B1 and zearalenone and were tested on human hepatocarcinoma cells with no increase in cytotoxicity observed. Analysis of treated maize revealed substantial changes to small molecular mass components of the matrix. While CAPP shows promise in terms of mycotoxin detoxification important questions concerning potential changes to the nutritional and safety status of the food matrix require further investigations.

Control of Fusarium verticillioides (Sacc.) Nirenberg and Fumonisins by Using a Combination of Crop Protection Products and Fertilization.

Fusarium verticillioides is the most common fungal pathogen associated with maize ear rot in Tanzania. In a two-year trial, we investigated the efficacy of crop protection (insecticide and/or fungicide) and fertilizer (nitrogen and/or phosphorus) treatments in reducing the occurrence of F. verticillioides and its mycotoxins in maize grown in Tanzania. Seasonal differences were seen to have a substantial influence on the incidence and severity of insect infestation, Fusarium ear and kernel rot, biomass of F. verticillioides and contamination with fumonisins. With regard to the application of fertilizers, it was concluded that the impact on maize stalk borer injury, Fusarium symptoms and fumonisin levels was not significant, whereas crop protection significantly reduced maize damage. The application of an insecticide was most effective in reducing insect injury and as a result of the reduced insect injury the insecticide treatment also resulted in a significant decrease in Fusarium symptoms. In 2014, fumonisin levels were also significantly lower in maize treated with an insecticide. Additionally, significant positive correlations between insect damage and Fusarium symptoms were observed. In conclusion, this study clearly shows that application of an insecticide alone or in combination with a fungicide at anthesis significantly reduces insect damage and consequently reduces F. verticillioides infection and associated fumonisin contamination.

Risk of Fungi Associated with Aflatoxin and Fumonisin in Medicinal Herbal Products in the Kenyan Market.

Utilization of herbal products is a major concern due to the possibility of contamination by toxigenic fungi that are mycotoxin producers such as Aspergillus species during processing and packaging. Research was carried out to determine the presence of aflatoxins and fumonisins in herbal medicinal products sold in Eldoret and Mombasa towns in Kenya. The study employed both exploratory and laboratory experimental design. The herbal products were purchased from the market and transported to Kenya Medical Research Institute for processing and analysis. Fungal contaminants were determined according to Pharmacopoeia specifications. The toxins were quantified using ELISA based technique. The genus Aspergillus was the most dominant followed by Penicillium. Fungal counts ranged between 1 CFU/g and >1000 cfu/g. Analysis of variance showed that the rate of fungal contaminants for Eldoret and Mombasa samples had significant association (p ≤ 0.001). Aflatoxin levels ranged from 1 to 24 ppb, while fumonisin levels ranged from 1 to >20 ppb. Only 31% of samples met the standards for microbial limits as specified in Pharmacopoeia. There is need for product microbial quality improvement through proper harvesting, processing, storage, and marketing. It is recommended that a policy be enacted to enable regulation of herbal products in Kenya.

Detection of Fumonisins in Fresh and Dehydrated Commercial Garlic.

An epidemic fungal disease caused by Fusarium proliferatum, responsible for fumonisin production (FB1, FB2, and FB3), has been reported in the main garlic-producing countries in recent years. Fumonisins are a group of structurally related toxic metabolites produced by this pathogen. The aim of this work was to establish an enzyme-linked immunosorbent assay (ELISA) procedure, mostly applied to cereals, that is suitable for fumonisin detection in garlic and compare these results to those obtained by high-performance liquid chromatography (HPLC) and screening of fresh and dehydrated garlic for toxicological risk. The results show good correlation between the two analytical methods. In fresh symptomatic garlic, fumonisin levels were higher in the basal plates than those in the portions with necrotic spots. Among the 56 commercially dehydrated garlic samples screened, three were positive by ELISA test and only one was above the limit of quantitation. The same samples analyzed by HPLC showed the presence of FB1 in trace amounts that was below the limit of quantitation; FB2 and FB3 were absent. The results are reassuring, because no substantial contamination by fumonisins was found in commercial garlic.

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

OBJECTIVE: To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. RESULTS: Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml-1 at 15 min of assay duration. CONCLUSIONS: The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Magneto-controlled aptasensor for simultaneous electrochemical detection of dual mycotoxins in maize using metal sulfide quantum dots coated silica as labels.

Currently there is an urgent need for multi-mycotoxin detection methods due to the co-occurrence of multiple mycotoxins in food raw materials and their augmented toxicity. Herein, a magneto-controlled aptasensor has been developed for simultaneous electrochemical detection of ochratoxin A (OTA) and fumonisin B1 (FB1), two typical mycotoxins found in food crops world-wide. This aptasensor was designed using the high specificity between the target and aptamer with heavy CdTe or PbS quantum dots (QDs) coated silica as labels and the complementary DNA functionalized magnetic beads as capture probes. In presence of targets, the aptamer preferred to form the target-aptamer binding which forced the partial release of the preloaded labels from the magnetic beads. After a one-step incubation and a simple magnetic separation, the electrochemical signals of Cd2+ and Pb2+ dissolved from the reserved labels which had negative correlation with targets contents, was measured based on the difference of peak potentials. This aptasensor provided a wide detection range of 10pgmL-1 to 10ngmL-1 for OTA and 50pgmL-1 to 50ngmL-1 for FB1, and succeeded in real maize samples. This method provides a new avenue for high throughput screen of mycotoxins due to the advantages of simple instrument, low sample consumption, short assay times, and lower detection costs per assay. Moreover, it could be readily expanded for the simultaneous detection of a large panel of mycotoxins by using different metal sulfide QDs when their specific aptamers are available.

Fumonisins B1 and B2 in the corn-milling process and corn-based products, and evaluation of estimated daily intake.

The distribution of fumonisins (FBs: FB1 and FB2) in the corn-milling process and in corn-based products, as well as daily intake estimates for the Brazilian population were evaluated. Among corn fractions samples, corn meal had the highest mean concentration of FB1 (1305 µg kg(-1)) and FB2 (651 µg kg(-1)) and a distribution factors of 452% and 256% in relation to corn grain, respectively. On the other hand, the distribution factor of FB1 and FB2 in corn flour was found to be 144% and 88% respectively, which demonstrates that fumonisins in this fraction were reduced compared with corn grain. As a result, almost half the corn meal samples (47%) would be non-compliant with future Brazilian regulation (2017) for fumonisins. However, corn-based products, such as corn flakes and popcorn, were in compliance with the regulation. The average probable daily intake and maximum probable daily intake of fumonisins estimated for the Santa Catarina state (Brazil) population were below the provisional maximum tolerable daily intake of 2 µg kg(-1) body weight day(-1) for all corn samples. Despite this, the adoption of practices to control the occurrence of fumonisins should be applied to the corn-milling fractions that may contain a higher concentration of this toxin, such as corn meal, often used for animal feed in Brazil.

Fumonisins B1, B2 and B3 in corn products, wheat flour and corn oil marketed in Shandong province of China.

In this study a total of 522 samples were collected from Shandong province of China in 2014 and analysed for the occurrence of fumonisin B1 (FB1), FB2 and FB3 by isotope dilution ultrahigh performance liquid chromatography-tandem mass spectrometry. Fumonisins were detected in 98.1% of the corn products, with the average total level of 369.2 µg kg(-1). The individual average values of FB1, FB2 and FB3 in corn products were 268.3, 53.7 and 47.2 µg kg(-1), respectively. The simultaneous occurrence of FB1, FB2 and FB3 was observed in 76.7% of the corn products. Especially, the results demonstrated that the difference in the contamination levels for fumonisins in these three types of corn products was apparent. In addition, 6.2% of the wheat flour samples were contaminated with FB1, with concentrations ranging from 0.3 to 34.6 µg kg(-1). No FB2 or FB3 was detected in wheat flour. In corn oil samples no fumonisins were detected.

UHPLC-MS/MS determination of ochratoxin A and fumonisins in coffee using QuEChERS extraction combined with mixed-mode SPE purification.

A method was developed for simultaneous determination of the mycotoxins: ochratoxin A (OTA) and fumonisins B2 (FB2), B4 (FB4), and B6 (FB6) in green, roasted, and instant coffee. Extraction was performed by QuEChERS (quick, easy, cheap, effective, rugged, and safe) under acidic conditions followed by mixed-mode reversed phase-anion exchange solid phase extraction. OTA and FB2 were detected at levels down to 0.5 and 2 µg/kg by UHPLC-MS/MS and quantitated via isotope dilution using U-(13)C-labeled FB2 and OTA as internal standards. Mixing 20% isopropanol in the acetonitrile of the acidic UHPLC gradient system increased the signal intensity by 50% and decreased the ion-suppression with 50-75% in roasted coffee samples. About half of the roasted coffee samples (n = 57, from 9 countries) contained detectable levels of OTA, however, with only 5 samples above the EU regulatory limit of 5 µg/kg and the highest with 21 µg/kg. None of the 25 instant coffee samples contained OTA above the EU regulatory level of 10 µg/kg. Nonetheless, the toxin could be detected in 56% of the analyzed instant coffee samples. Fumonisins were not detected in any of the roasted or instant coffee samples (n = 82). However, in the green coffee samples (n = 18) almost half of the samples were positive with a maximum value of 164 µg/kg (sum of FB2, FB4, and FB6). This discrepancy between green coffee and processed coffees indicated that the fumonisins decompose during the roasting process, which was confirmed in roasting experiments. Here fumonisins could not be detected after roasting of the green, 164 µg/kg coffee, sample. Under the same conditions, OTA was reduced from 2.4 to 0.5 µg/kg.

Host extract modulates metabolism and fumonisin biosynthesis by the plant-pathogenic fungus Fusarium proliferatum.

Fusarium proliferatum is a common pathogen able to infect a broad range of agriculturally important crops. Recently, some evidence for genetic variance among the species genotypes in relation to their plant origin has been reported. Mycotoxin contamination of plant tissues is the most important threat caused by F. proliferatum and fumonisins B (FBs) are the principal mycotoxins synthesized. The toxigenic potential of the pathogen genotypes is variable and also the reaction of different host plant species on the infection by pathogen is different. The objective of present study was to evaluate the impact of the extracts on the growth and fumonisin biosynthesis by 32 F. proliferatum strains originating from different host species (A-asparagus, M-maize, G-garlic, PS-pea and P-pineapple), and how it changes the secondary metabolism measured by fumonisin biosynthesis. The average strain dry weight was 65.2 mg for control conditions and it reached 180.7 mg, 100.5 mg, 76.6 mg, 126.2 mg and 51.1 mg when pineapple, asparagus, maize, garlic and pea extracts were added, respectively. In the second experiment the extracts were added after 5 days of culturing of the representative group of strains, displaying diverse reaction to the extract presence. Also, the influence of stationary vs. shaken culture was examined. Mean biomass amounts for shaken cultures of 15 chosen strains were as follows: 37.4 mg of dry weight for control culture (C), 219.6 mg (P), 113 mg (A), 93.6 mg (M), 62 mg (G) and 48 mg (PS), respectively. For stationary cultures, the means were as follows: C-57.4 mg, P-355.6 mg, A-291.6 mg, M-191.1 mg, G-171.1 mg and PS-58.9 mg. Few strains showed differential growth when stationary/shaken culture conditions were applied. Almost all strains synthesized moderate amounts of fumonisins in control conditions-less than 10 ng/µL, regardless of the origin and host species. Few strains were able to produce over 100 ng/µL of FBs when pineapple extract was added, twelve strains synthesized more than 10 ng/µL under asparagus extract induction and the pea extract was the most efficient inhibitor of fumonisin biosynthesis. The general impact of the extracts on the fungal biomass amounts was similar, regardless of the host plant origin of the fungal genotypes studied. The evaluation of FBs content has shown differential reaction of some strains, which may contribute to their aggressiveness and pathogenicity.

Simultaneous detection of ochratoxin A and fumonisin B1 in cereal samples using an aptamer-photonic crystal encoded suspension array.

A simple, new aptamer-photonic crystal encoded suspension array was designed to simultaneously quantify and qualify ochratoxin A(OTA) and fumonisin B1(FB1) in cereal samples. The aptamers of OTA and FB1 were immobilized on the surfaces of photonic crystals by chemical bonding. When the target mycotoxins appear in a sample, the fluorescence-labeled complementary DNA of the aptamer dissociates from their double DNA hybrid and results in an obvious decrease in fluorescence intensity of the microsphere. The difference value of fluorescent intensities for each kind of silica photonic crystal microsphere (SPCM) quantitatively conveys the concentration of mycotoxin, and the structure colors or reflectance peak positions of the SPCMs confirm the kind of mycotoxin detected. The reaction conditions including the immobilization method for aptamers, hybridization, and incubation conditions have been optimized. This developed method displayed a wide linear detection range (0.01-1 ng/mL for OTA and 0.001-1 ng/mL for FB1) and a low limit of detection (0.25 pg/mL for OTA and 0.16 pg/mL for FB1). The recovery rates in the spiked cereal samples ranged from 81.80% to 116.38% for OTA and 76.58%-114.79% for FB1. The positive detection results in the naturally contaminated cereal samples were in agreement with those of classic enzyme-linked immunosorbent assay (ELISA). This simple suspension array scheme displays a great application potential for the high throughput screen assay of mycotoxins.

Bioguided isolation, characterization, and biotransformation by Fusarium verticillioides of maize kernel compounds that inhibit fumonisin production.

Fusarium verticillioides infects maize ears, causing ear rot disease and contamination of grain with fumonisin mycotoxins. This contamination can be reduced by the presence of bioactive compounds in kernels that are able to inhibit fumonisin biosynthesis. To identify such compounds, we used kernels from a maize genotype with moderate susceptibility to F. verticillioides, harvested at the milk-dough stage (i.e., when fumonisin production initiates in planta), and applied a bioguided fractionation approach. Chlorogenic acid was the most abundant compound in the purified active fraction and its contribution to fumonisin inhibitory activity was up to 70%. Moreover, using a set of maize genotypes with different levels of susceptibility, chlorogenic acid was shown to be significantly higher in immature kernels of the moderately susceptible group. Altogether, our data indicate that chlorogenic acid may considerably contribute to either maize resistance to Fusarium ear rot, fumonisin accumulation, or both. We further investigated the mechanisms involved in the inhibition of fumonisin production by chlorogenic acid and one of its hydrolyzed products, caffeic acid, by following their metabolic fate in supplemented F. verticillioides broths. Our data indicate that F. verticillioides was able to biotransform these phenolic compounds and that the resulting products can contribute to their inhibitory activity.

Use of liquid chromatography-high-resolution mass spectrometry for isolation and characterization of hydrolyzed fumonisins and relevant analysis in maize-based products.

The synthesis of partially hydrolyzed fumonisins (PHFB1 and PHFB2) and hydrolyzed fumonisins (HFB1 and HFB2) by chemical hydrolysis of pure fumonisins (FB1 and FB2) is reported together with the isolation and characterization by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Two structural isomers of partially hydrolyzed forms of FB1 and FB2 were identified, namely PHFB(1a) and PHFB(1b) and PHFB(2a) and PHFB(2b). Reaction yields were 21% for PHFB1 (sum of the two isomers), 52% for HFB1, 31% for PHFB2 (sum of the two isomers) and 30% for HFB2. Purity of each isolated compound was >98%. An LC-HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzed derivatives was applied to 24 naturally contaminated samples of maize and maize-based products. The majority of samples (18 out of 24) were contaminated with fumonisins B1 and B2. Fumonisins co-occurred with both partially hydrolyzed and hydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co-occurrence of fumonisins with partially hydrolyzed fumonisins was also recorded in one sample of maize kernels and four samples of maize-based products (i.e. maize meal, cous-cous, corn-cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 µg/kg for fumonisins (sum of FB1 and FB2), from 10 to 210 µg/kg for partially hydrolyzed fumonisins (sum of PHFB1 and PHFB2) and from 30 to 200 µg/kg for hydrolyzed fumonisins (sum of HFB1 and HFB2). This is the first report of the isolation of PHFB2 and the co-occurrence of FB1, FB2, PHFB1, PHFB2, HFB1 and HFB2 in maize products. Considering the growing use of nixtamalized and maize-based products, the monitoring of fumonisins and their partially and totally hydrolyzed forms in these products may represent an important contributing factor in evaluating the relevant human risk exposure.