Einfluss der lagerdauer und des feuchtegehaltes auf die bildung von ochratoxin A und citrinin bei körnerleguminosen aus ökologischem und konventionellem anbau

Influence of storage time and moisture content on the development of ochratoxin A and citrinin in legumes kernel of ecological and conventional provenance Mould growth can cause the occurrence of mycotoxins in grain and legumes. Less information is known for legumes of ecological provenance. For this reason a storage trial was carried out with peas and horse beans, to examine the production of ochratoxin A (OTA) and citrinin (CT) in legumes kernel from ecological provenance. For that purpose kernels from legumes were remoistened to different moisture contents (MC, 14%/19%) and stored 24 weeks in a research granary (tower silo). This experiment should simulate the storage situation in farm scale from winter to summer. Every four weeks, the CO₂-content was determined and samples taken for the analysis of moisture, OTA and CT. At week 24 and a MC of <18% 1.9 μg OTA/kg of beans and 0.7 μg OTA/kg of peas (conventionally produced) were found.

Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Detection of citrinin in ochratoxin A-containing products by a new HPLC method

A new method for citrinin was developed and validated, which is based on solid phase extraction with polyamide columns and HPLC with fluorescence detection. Sufficient skill with the method given, precise results, i.e. variation coefficients <10%, will be achieved. The mean recovery rates were in the range 74 – 90%. The detection limits of the method determined according to DIN 32645, at good precision, were 1 μg/kg for wheat, rye, barley, maize, and oats. The analysis of several samples containing ochratoxin A (OTA) showed that citrinin is present in brans, wheatings and shorts containing a higher ratio of the outer layers of the grain kernel; both OTA and citrinin were found in in cocoa shells and raisins. Citrinin was detected in 14 OTA-containing samples (1–8 μg/kg). Furthermore, it was demonstrated that citrinin also can be determined in red mold rice according to the new method. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Natural occurrence of ochratoxin A and citrinin in food stuffs in Egypt

Natural occurrence of ochratoxin A (OA) and citrinin in cereals (274 samples) and animal tissues (250 samples) have been investigated during a period of more than 2 years. OA was found in cereals and animal tissues while citrinin was found in cereals only. The highest level of OA (up to 80.0 μg/kg) was found in yellow corn, 52.8% of contaminated samples while respectively 55.9% and 39.4% of barley and rice samples were contaminated with citrinin, with the highest level up to 100.0 and 27.92 μg/kg for barley and rice respectively. The frequent contamination of animal kidney with OA (28% positive out of 150 tested) average concentration 12.33 μg/kg. 2% of liver and 4% of muscles tissue were observed.     

Anwendung der automatisierten Festphasenextraktion bei der Bestimmung von Ochratoxin A

For some foodstuffs, determination of the mycotoxin ochratoxin A (OTA) requires time consuming clean up by means of solid phase extraction (SPE). Therefore a system for automated SPE was tested for cleaning up roasted coffee as a possible way of shortening preparation time. Validation of the method in accordance to the so called “Concept '98” led to a LOD of 0.2 μg/kg and a recovery rate of 92%. By using the described procedure with samples of roasted coffee the OTA contents varied between the LOD and 3.4 μg/kg. This method was also used to determine ochratoxin A in liquorice roots, ginger and valerian.

Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Citrinin,ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy

Experiments with Neisseria meningitidis have shown that Fe³⁺ to some extent can reverse the toxicity of ochratoxin A and citrinin, as measured by inhibition zones around impregnated paper discs. Similar phenomena were observed with the less toxic ochratoxin B. Zearalenone also inhibited growth, but its effect was not counteracted by iron. The mycotoxins aflatoxin B₁ and deoxynivalenol did not inhibit bacterial growth at all. Desferal (deferoxamine) also inhibited growth of meningococci, but iron totally abolished this inhibition. The results indicate that ochratoxin A and citrinin interfere with iron metabolism in this organism but that other additional toxic mechanisms are involved as well since a marked growth inhibition by both toxins was also observed in the presence of iron. One function of ochratoxin A and citrinin in nature could consequently be to affect the iron uptake of other competing microorgansms.Since both toxins interfere with iron and both cause nephropathy, a possible connection between these properties and lipid peroxidation is also briefly discussed.Abbreviations DON deoxynivalenol - OA ochratoxin A - OB ochratoxin B     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

Mycobiota of ground red pepper and their aflatoxigenic potential

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 10³ CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03?2.08 μg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88?909.53 μg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.     

Conversion of the mycotoxin citrinin into dihydrocitrinone and ochratoxin A by Penicillium viridicatum

Summary The mycotoxin citrinin, produced by Penicillium viridicatum, was found to be unstable in broth culture. This instability was investigated using the techniques of ¹⁴C-labelling, mass spectrometry and thin layer chromatography. It was concluded that intracellular factors released by P. viridicatum were responsible for the instability of the mycotoxin. The major products resulting from the breakdown of citrinin were identified as 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethyl-isocoumarin-7-carboxylic acid (dihydrocitrinone) and ochratoxin A.     

Untersuchungen Zur Kühllagerung Von Weizen

Abstract

Saatweizen wurde ohne vorhergchende Sterilisation mit einem Ochratoxin A und Citrinin bildenden Stamm von Penicillium verrucosum beimpft und bei Wassergehalten von 18, 20, 22, 24 und 26% bei 4 und 10°C gelagert. Die Produktion von Ergosterin, eines chemischen Indikators für die von Pilzen gebildete Biomasse, setzte innerhalb der untersuchten Lagerdauer (240 Tage) ein. Lediglich bei 18% H₂O/4°C war keine Zunahme des Ergosteringehaltes zu beobachten. Ochratoxin A und Citrinin waren bei 18% H₂O/4°C und 20% H₂O/4°C über 240 Tage nicht zu finden (Nachweisgrenze: 10 bzw. 25 μg/kg). Bei den übrigen Kombinationen von Wassergehalt und Temperatur begann die Anhäufung der beiden Toxine etwa gleichzeitig mit der Ergosterinproduktion. Mit steigendem Wassergehalt und steigender Temperatur nahm die Zeit bis zum Beginn der Ergosterinproduktion ab, während die Geschwindigkeit der Produktion von Ergosterin, Ochratoxin A und Citrinin zunahm. Beide Toxine wurden während einer ersten Phase der Anhäufung etwa mit gleicher Rate gebildet. Bei 20–26% H₂O hatten der Wassergehalt und die Temperatur keinen Einfluß auf die Beziehung zwischen dem Toxingehalt und dem gleichzeitig erreichten Ergosteringehalt. Es wird empfohlen, stark mit Penicillium verrucosum kontaminierten Weizen nicht über den Beginn der Ergosterinproduktion hinaus zu lagem.

INVESTIGATIONS ON REFRIGERATED STORAGE OF WHEAT

1. Ergosterol, ochratoxin A and citrinin after inoculation with Penicillium verrucosum

Seed wheat was innoculated without having been sterilized with an ochratoxin A and citrinin forming strain of Penicillium verrucosum and stored at moisture contents of 18, 20, 22, 24, and 26% at 10 and 4°C. The production of ergosterol, a chemical indicator of fungal biomass, started within the storage time investigated (240 days). Only at 18% H₂O/4°C an increase of the ergosterol content was not observed. Ochratoxin A and Citrinin were not detected at 18% H₂O/4°C and 20% H₂O/4°C within 240 days (detection limit: 10 and 25 μg/kg, respectively). At the other combinations of moisture content and temperature the first detection of the two toxins approximately coincided with the onset of ergosterol production. With increasing moisture content and temperature the time up to the start of ergosterol production decreased, whereas the production rates of ergosterol, ochratoxin A and citrinin increased. Both toxins were produced with about the same rate during a first phase of accumulation. At 20–26% H₂O there was no influence of moisture content and temperature on the relation between toxin content and the simultaneously reached ergosterol content. It is recommended that wheat highly contaminated with Penicillium verrucosum should not be stored beyond the start of ergosterol production.     

Fate of Ochratoxin A and Citrinin During Malting and Brewing Experiments

The fate of ochratoxin A and citrinin during malting and brewing processes was studied by the use of naturally contaminated lots of barley, as well as by the addition of crystalline toxins to the mash. Complete degradation was observed for ochratoxin A from moderately contaminated barley lots and for citrinin added to mash. The use of highly contaminated barley resulted in transmission of ochratoxin A into the beer, but only 2 to 7% of the initial content was detected, corresponding to levels of 6 to 20 mug of ochratoxin A per liter of beer. Barley lots with this high ochratoxin contamination (1,000 to 5,000 mug/kg) will be easily detected and, therefore, because of pronounced deterioration, should be rejected during inspection upon admittance to the breweries.     

Natural occurrence of mycotoxins in different spices in Egypt

A total of 120 different samples belonging to 24 kinds of spices collected from different places atAssiut Governorate (Egypt) were examined for the natural occurrence of mycotoxins. TLC analysis of spice extracts revealed the presence of aflatoxins (8–35 μg/kg) in 16 samples of anise, black pepper, caraway, black cumin, fennel, peppermint, coriander and marjoram, sterigmatocystin (10–23 μg/kg) in ten samples of red pepper, caraway, cumin and marjoram and citrinin (8–12 ⧎g/kg) in two samples of black cumin, while ochratoxin A and zearalenone could not be detected.     

Feeding OTA to pigs: Analytical evaluation of a trial

Methods were developed to analyse the concentrations of ochratoxin A (OTA) and its main metabolite ochratoxin α in blood plasma of pigs and in their kidneys. By the use of these methods blood and kidneys of pigs that were fed contaminated feedstuff containing 1500 μg/kg ochratoxin A for the whole trial period of 42 days were analysed.High levels of 1500 – 2000 μg/I OTA were found in the plasma, while the concentration of OTA in the kidneys did not exceed 250 μg/kg at the end of the trial. Neither in the plasma nor in the kidneys of the animals fed contaminated feedstuff ochratoxin a could be detected.     

Occurrence of ochratoxin A in herbal drugs of Indian origin — a report

This paper contains a report of occurrence of ochratoxin A in some common herbal medicines collected from different store-houses and shop-keepers of Bihar, India. Of 129 samples of 9 plants, 55 were found to be contaminated with various levels of ochratoxin A. The level of ochratoxin A was found maximal in barks ofHolarrhena antidysenterica (1.14 – 2.34 μg/g) whereas it was minimal in rhizomes ofTacca aspera (0.3 – 0.74 μg/g).Aspergillus ochraceus, A sulphureus and Penicillium viridicatum isolates obtained from drug samples were also examined for their toxigenic potentials. 19 isolates ofA ochraceus, 13 ofA sulphureus and 37 isolates ofP viridicatum were found to be toxigenic out of 67, 33, and 107 isolates, respectively. The ochratoxin A produced by Aochraceus was in the range of 0.09 to 2.44 μg/mL, byA sulphureus 0.1 to 1.76 μg/mL, and byP viridicatum 0.14 to 2.78 μg/mL of the culture filtrate.     

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.     

Evaluation of fungal contamination and mycotoxin production in maize silage

Agricultural activities involve daily use of maize silage as feed for livestock, which can be contaminated by mycotoxigenic molds. To evaluate fungal contamination, and the production of mycotoxins in maize silage we propose a multi-disciplinary approach utilizing PCR methods with genes of the aflatoxin (ver-1, omt-1 and apa-2), fumonisin (FUM1) and trichothecene (TRI6) biosynthesis pathways. To detect Aspergillus fumigatus, a 26S/intergenic spacer region of the rDNA complex was amplified. These specific PCR assays allowed three major groups of toxigenic fungi-like aflatoxin-producing Aspergilli, fumonisin and trichothecene-producing Fusaria, and the ubiquitous mold A. fumigatus, to be targeted. A multimycotoxin method is also proposed to simultaneously quantify seven mycotoxins (i.e., aflatoxin B₁, citrinin, deoxynivalenol, fumonisin B₁, gliotoxin, ochratoxin A, zearalenone) in maize silage by high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS). These microbiological and analytical tools revealed three potentially toxigenic groups of fungi and A. fumigatus grown from mature maize silage (11 month old) that was collected in Normandy (France) and the mycotoxins aflatoxin B₁ (7.0–51.3 μg/kg), citrinin (10.1–14.2 μg/kg), deoxynivalenol (128.0–181.0 μg/kg) and gliotoxin (6.6–11.9 μg/kg). Results indicate that the combination of PCR and HPLC–MS can be used to assess fungal quality of maize silages.     

Biotransformation of plantain pseudostem fibres using local enzyme sources; analysis of their potential as commercial poultry feed

Plantain pseudo-stem fibres (PPS) were valorized in this study by subjecting to simultaneous saccharification and fermentation (SSF) and afterwards, their complex carbohydrates and monosaccharides, mycotoxins, protein qualities, and free radical scavenging potentials were compared to those of commercial poultry feeds (CPF). The SSF of PPS was achieved using digestive juice of the snail; Archachatina marginata, and yeast, while standard methods like HPLC-UV, HPLC-DAD, monosaccharides and mycotoxin kits, and UV-VIS spectrophotometry were used for analysis. The cellulose, hemicellulose, pectin, lignin, extractives, and acetyl contents of PPS were significantly (p?<?.05) reduced when subjected to SSF. Glucose (41.1%), galactose (11.2%), mannose (1.7%), and fucose (1.8%) contents of the SSF-PPS were higher than those of the PPS and CPF while CPF showed higher contents of arabinose (8.2%), fructose (18.3%), and rhamnose (1.7%). No mycotoxin was detected in the PPS, while all aflatoxins (B₁, B₂, G₁, and G₂), citrinin, fumonisin B₁, and B₂, ochratoxin A and B contents of SSF-PPS were equivalent to those for CPF. Patulin (5.52×10^?4?µg/kg) and zearalenone (7.76×10^?6?µg/kg) contents of the SSF-PPS were lower than those for CPF (1.50×10^?3?µg/kg and 1.13×10^?5?µg/kg respectively). The total amino acids (TAA), total non-essential and essential amino acids (TNEAA and TEAA), total basic and branched chain amino acids (TBAA and TBCAA) of the SSF-PPS were higher than those of the PPS and CPF while the free radical scavenging potentials of the SSF-PPS were mostly concentration dependent, and showed significantly higher ABTS, DPPH, Ferric, OH, lipid peroxide, and superoxide radical scavenging potentials than the standards used. This study has shown that the valorization of the agricultural residue using SSF, improves carbohydrate, protein, mycotoxins, and in-vitro antioxidant properties suitable enough for poultry feeding.     

Antifungal properties of selected lactic acid bacteria and application in the biological control of ochratoxin A producing fungi during cocoa fermentation

Thirteen Lactic acid bacteria strains isolated from fermenting cocoa and seven reference strains were used in order to assess their antifungal properties towards three ochratoxin A (OTA) producing fungi (Aspergillus carbonarius, Aspergillus niger and Aspergillus ochraceus). Furthermore, two of the isolates strains (A19 and A21) identified as belonging to the genus of Pediococcus as well as Lactobacillus plantarum B4496, Lactobacillus brevis 207 and Lactobacillus sanfranciscensis BB12 showed interesting in vitro broad antifungal activities towards the three ochratoxin-producing fungi with inhibition percentages ranging from 15% to 66.7%. Treatment of cell-free supernatant at 100°C affected antifungal activity suggesting that the main compounds responsible for this activity were of proteic nature, and hence could be bacteriocins. Application of isolate A19 in cocoa fermentation as starter inhibited the growth of each of the OTA-producing species. At the end of fermentation in boxes inoculated with A19, A. niger was not detectable while A. carbonarius concentration was found to be 2 Log CFU/g of wet beans. The assessment of the ochratoxin produced during fermentation of cocoa inoculated with A. carbonarius indicated that the use of isolate A19 as starter could reduce their level of growth so as to have only a toxin production of 0.0012 ± 0.0005 μg/kg after 40 days of storage, while this was 2.45 ± 0.35 μg/kg of fermented and dried cocoa beans in the absence of A19. This work is a contribution for the application of biological control of OTA-producing fungi during cocoa production.     

Production of ochratoxin a,citrinin, and ergosterol byPenicillium viridicatum in autoclaved and non-autoclaved wheat at low temperature

Wheat (moisture content: 26%) was autoclaved or left untreated, inoculated with conidia ofPenicillium viridicatum and stored at 10°C. The fungus grew on both substrates and was the dominant mould on the non-autoclaved grain. Autoclaving resulted in an earlier onset of ergosterol, ochratoxin A, and citrinin production due to accelerated mould growth. Yield of ochratoxin A increased while citrinin slightly decreased in autoclaved wheat.     

Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing penicillia.

In a batch of barley associated with field cases of mycotoxic porcine nephropathy and containing ochratoxin A and citrinin, the mycoflora were isolated by parallel incubation at 10 and 25 degrees C. Subsequently, the isolated cultures were checked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin, and citrinin). The nephrotoxin producers, all isolated by incubation at 10 degrees C, were comprised of one culture of Penicillium viridicatum, five cultures of Penicillium cyclopium, and one culture of Penicillium crustosum, all producing xanthomegnin and viomellein. One culture of P. cyclopium produced citrinin. Viomellein was detected in the barley at a concentration of approximately 1 mg/kg. The method of analysis for xanthomegnin and viomellein included extraction with chloroform, partitioning in hexane-acetone, and thin-layer chromatographic separation and identification. The identity of the xanthomegnin and viomellein produced by the isolated fungi and of viomellein detected in the barley was supported by infrared spectroscopy. This is the first report of viomellein as a natural contaminant of foodstuffs.     

Low occurrence of patulin‐ and citrinin‐producing species isolated from grapes

Aims: To assess the ability of fungi isolated from grapes to produce patulin and citrinin. Methods and Results: A total of 446 Aspergillus isolates belonging to 20 species and 101 Penicillium isolates were inoculated in Czapek yeast extract agar and yeast extract sucrose agar and incubated for 7 days at 25°C. Extracts were analysed for patulin and citrinin by thin‐layer chromatography. None of the isolates of Aspergillus spp. produced either patulin or citrinin. Patulin was produced by three isolates of Penicillium expansum and two of Penicillium griseofulvum. Citrinin was produced by five isolates of P. expansum, two of Penicillium citrinum and one of Penicillium verrucosum. Conclusions: Our results show that the Aspergillus and Penicillium species commonly isolated from grapes are not a source of the mycotoxins, patulin and citrinin. Significance and Impact of the Study: The possibility of co‐occurrence of patulin and citrinin with ochratoxin A in grapes and grape products remain low, owing to the low frequency of isolation of potentially producing species.