Johannes Krämer

Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters

The occurrence of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA) in the winter wheat of 1997 and 1998 grown under organic farming conditions was investigated using ELISAs (R-Biopharm®) for quantification. The influence of delayed drying of the grain after harvest on the development of DON and OTA was determined in storage trials (moisture: 17% and 20%; temperature: 20°C; duration: four and six weeks). The Tox5 PCR assay was used both to detect Fusarium species with the potential to produce trichothecenes and as a measure of their relative DNA content during the storage trials. The intensity of the PCR signals was correlated with the DON concentration. Fusarium species were identified microscopically by standard methods. All the freshly harvested grain samples were contaminated with DON and showed further increases in the DON concentration during storage. OTA contamination was found in 14.3% of the 1997 samples and in 24.1% of the 1998 samples. OTA increased during storage trials of the 1997 samples but not in the 1998 samples.

Epidemiology of Fusarium Infection and Deoxynivalenol Content in Winter Wheat in the Rhineland, Germany

Details of our long-term research programme concerning the epidemiology of Fusarium spp. and mycotoxin production are summarized. Evaluation of the occurrence of Fusarium spp., mainly on winter wheat (Triticum aestivum), was carried out by investigating Fusarium infection and mycotoxin contamination. Two to 15% of grains were infested during 1995–1998 at three climatologically differing localities of the Rhineland, Germany. Disease progress was accelerated by rainfall during the flowering season. The species most frequently isolated were Fusarium avenaceum, F. poae, F. culmorum and F. graminearum. The mean deoxynivalenol (DON) content varied from 19μgkg−1 (1995) to 310μgkg−1 (1998) and was not always correlated with disease severity. Organic farming systems showed lower rates of infection with ear blight and lower mycotoxin contamination than conventional farming systems.

Thermal inactivation of poliovirus type 1 in water, milk and yoghurt

Loss of infectivity of poliovirus type 1, strain Sabin, during heating, freezing, and storage in water, milk and yoghurt was determined by plaque-titration in Vero cell cultures. The heating experiments simulated the conditions arising during the processing of milk and yoghurt, for example high-temperature heating (95 degrees C, 15 and 30 s), short-time pasteurization (72 degrees C, 15 and 30 s), long-time pasteurization (62 degrees C, 30 min), and yoghurt-fermentation (42 degrees C, 30 min and 180 min). Only high-temperature heating, long-time pasteurization and short-time pasteurization for 30 s proved to be reliable methods of inactivating polioviruses present in water, milk and yoghurt completely. Short-time pasteurization for 15 s and the conditions of yoghurt-fermentation failed to cause complete inactivation of polioviruses. Additionally, polioviruses mixed in milk or yoghurt withstood these procedures with significantly lower reductions of infectivity than in water. Heating at 55 degrees C for 30 min resulted in complete inactivation of polioviruses, regardless of the suspending medium. The infectivity of polioviruses is scarcely affected by freezing (-20 degrees C, 30 min) and storage (24 days) at low temperatures (4 degrees C) and high humidity (a(w) = 0.99).

Inactivation of staphylococcal enterotoxins by heat and reactivation by high pH treatment

Inactivation of staphylococcal enterotoxins (SE) added to different media upon heat treatment (80 degrees C or 100 degrees C for 10 min) and reactivation of inactivated SE was studied. In gelatin (pH 4.0), lettuce extract, peas and beans extracts and ovalbumin (pH 5.0) the immunological activity of SE was lost almost completely upon heating. The loss of immunological activity of SEA was accompanied by a concomitant loss of biological activity of this toxin (monkey feeding test). A high pH treatment (pH 11) of the different preparations restored both the immunological and biological activity in most samples tested. Heating at 80 degrees C or 100 degrees C for 10 min of SE containing gelatin (pH 7.0), cauliflower extract (pH 4.0 or pH 7.0), milk (pH 4.0), casein (pH 6.0), rice extract (pH 7.0), noodles extract (pH 4.0) and oat-flakes extract (pH 7.0) had a much lower effect on the immunological activity of the SE (activity greater than or equal to 25%).

Incidence ofFusaria and occurrence of selected Fusarium mycotoxins on Lolium spp. in Germany

Test plantings with varieties ofLolium multiflorum andL perenne were harvested 4 to 7 times a year in 1991 and 1992. Samples were checked for the presence ofFusaria, the mycotoxins zearalenone, T-2 toxin, and diacetoxyscirpenol (DAS). Spectrum of species and the incidence ofFusaria and fusariotoxins are discussed in relation to the influencing factors site, variety ofLolium, harvesting time and year. Depending on these factors, 41 % to 100 % of the samples wereFusarium positive. Differences in infestation with Fusarium among varieties ofLolium perenne were dependent on location and did not correlate with yield. The six species ofFusarium pathogenic toLolium spp. (F. graminearum, F. culmorum, F. avenaceum, F. oxysporum, F. solani, and F. acuminatum) totaled 35.7 % of all the isolated strains. 14 species could be isolated fromLolium samples (descending frequency):F. culmorum, F. sambucinum, F. equiseti, F. acuminatum, F. semitectum, F. oxysporum, F. subglutinans, F. avenaceum, F. sporotrichioides, F. proliferatum, F. tricinctum, F. anthophilum, F. dimerum and F. graminearum. For the detection ofFusaria a promising new immunological method is presented. It is based on the genus specific production of exopolysaccharides byFusarium species.Mycotoxin contents in grass ranged from 0.01 to 4.75 ppm for zearalenone with 67 % positive samples and 0.3 % samples above 1 ppm, 0.04 to 2.78 ppm for T-2 toxin with 25 % positive samples and 2.8 % samples above 1 ppm, and 0.003 to 0.06 for DAS with 21.6 % positive samples. In silages, no T-2 toxin was detectable. IsolatedFusarium strains were checked for the ability to produce the mycotoxins zearalenone, T-2 toxin and DAS in culture. Most of the strains were positive for at least one of the toxins.

Production of enterotoxins and thermonuclease by Staphylococcus aureus in cooked egg-noodles.

Production of enterotoxin A (SEA), enterotoxin C (SEC), and thermonuclease (TNase) by Staphylococcus aureus was determined during growth in cooked egg-noodles at different temperatures (15-37 degrees C). Both SEA and SEC and TNase were detected when greater than or equal to 4.0 x 10(7) colony forming units (cfu)/g were present. The contents of SEA, SEC, and TNase in egg-noodles mainly increased at the end of the exponential growth phase. In contrast with SEA and SEC the production of TNase always continued till the end of each experiment. Recovery rates of SEA and TNase in cooked noodles were dependent on their amounts. High amounts (64 ng SEA/g; 1 unit TNase/g) were recovered at a rate of 93% (SEA) and 54% (TNase) respectively, whereas low concentrations (1 ng SEA/g; 0.004 units TNase/g) were recovered at a rate of only 45% (SEA) and 1.1% (TNase). TNase usually is produced at all conditions which allow growth of S. aureus. Evidence of TNase was proposed for screening for staphylococcal enterotoxins (SE) in foods. But sometimes foods contain no TNase but SE. For this reason the ELISA-test which is simple and sensitive should be used for determination of SE-production in foods.

Pre-harvest strategies to ensure the microbiological safety of fruit and vegetables from manure-based production systems

Publisher Summary Nutritional quality is determined by the value of the product for the consumers physical health, growth, development, reproduction, and general well-being. Nutritional quality describes the inherent biological or health value of produce including the ratio of beneficial to harmful substances, taste, fragrance, freshness, and shelf-life, as well as the risk of pathogen contamination as important quality characteristics that govern consumer behavior. Traditional whole food recommendations as well as public information campaigns, such as “Five-a-Day for Better Health”, have recommended increased consumption of fresh (raw) fruit and vegetables in western diets. Higher per capita consumption of fresh or Minimally Processed Fruits and Vegetables [“MPF”], ready-to-eat produce, as well as the increase in imports of fresh fruits and vegetables from countries where hygiene standards may be low has resulted in heightened interest in outbreaks of human gastroenteritis, which may be attributed to contaminated fresh food, particularly vegetables.

Rheological and breadmaking properties of wheat samples infected withFusarium spp.

Rheological and breadmaking properties of untreated and suboptimally stored wheat samples (grain moisture: 20%, temperature: 20°C) and also of wheat which was inoculated withFusarium spp. were investigated. The deoxynivalenol (DON) content of the stored and inoculated wheat samples ranged between 820–12,000 μg/kg. Gluten proteins were isolated with different extraction solutions and the fractions obtained were analysed by means of RP-HPLC. Microextension tests and micro-baking tests were used for the determination of dough properties (maximum resistance (MR) and extensibility (EX)) and bread volume, respectively. In spite of the extremely high DON concentrations of some wheat samples contaminated withFusarium spp. they showed only a slight decrease of the amount of gluten proteins. Extension tests of dough led to a slight decrease of MR, bread volumes stayed almost the same compared with the non-contaminated grain. The contamination of wheat withAspergillus andPenicillium led to a high decrease of gluten proteins, which resulted in an extremely decreased MR of the dough and a very low bread volume.

Influence of water activity on the production of T-2 Toxin byFusarium sporotrichioides.

The production of T-2 Toxin by two strains ofFusarium sporotrichioides at high (0.995), medium (0.970) and low (0.945) water activity (aw) was investigated. The organisms were incubated in an optimal liquid base medium (Wort broth) adjusted to the different aw values. With decreasing aw the amount of T-2 Toxin in the culture supernatants fell drastically. Comparing cultures that had reached almost equal mycelial dry weights, those of low aw had T-2 Toxin levels 10 to 100 times lower than those of high aw. This effect was observed using both glycerol or sodium chloride as humectants.

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.