Development of an enzymatic assay for the quantitative determination of trypsin inhibitory activity in wheat.

Wheat is one of the world's most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.

A limited survey of aflatoxin B1 contamination in Indonesian palm kernel cake and copra meal sampled from batches.

Samples from large (100-200 tons) batches of palm kernel cake (PKC, n = 20) and copra meal (CM, n = 13) were collected at production facilities of four Indonesian feed mill manufacturers and analysed for aflatoxin B1 (AFB1) by ELISA. Recoveries using spiked samples ranged from 86 to 113%, with relative standard deviations of <9% (PKC) and <6% (CM). All batches were positive for AFB1: in PKC, at levels of 5.8-93.1 µg/kg (mean 49 µg/kg), and in CM, at levels of 1.1-147 µg/kg (mean 38.1 µg/kg). AFB1 levels were, in most batches, below the maximum level (100 µg/kg) recommended by the National Standardisation Agency, Republic of Indonesia. However, about half of the batches exceeded both the European Union and USA regulations for AFB1 in animal feed. In conclusion, serious efforts are necessary to control production, storage and shipment of palm kernel cake and copra meal for feed purposes, and clearly not only for products intended for export but also to reduce AFB1 levels in domestic Indonesian feed.

Uncertainty from sampling in measurements of aflatoxins in animal feedingstuffs: application of the Eurachem/CITAC guidelines.

The duplicate method for estimating uncertainty from measurement including sampling is presented in the Eurachem/CITAC guide. The applicability of this method as a tool for verifying sampling plans for mycotoxins was assessed in three case studies with aflatoxin B(1) in animal feedingstuffs. Aspects considered included strategies for obtaining samples from contaminated lots, assumptions about distributions, approaches for statistical analysis, log(10)-transformation of test data and applicability of uncertainty estimates. The results showed that when duplicate aggregate samples are formed by interpenetrating sampling, repeated measurements from a lot can be assumed to approximately follow a normal or lognormal distribution. Due to the large variation in toxin concentration between sampling targets and sometimes very large uncertainty arising from sampling and sample preparation (U(rel) ≥ 50%), estimation of uncertainty from log(10)-transformed data was found to be a more generally applicable approach than application of robust ANOVA.

Sol-gel immunoaffinity chromatography for the clean up of ochratoxin A contaminated grains.

This paper describes the application of sol-gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol-gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol-gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 µg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 µg/kg. Sol-gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.

Determination of ochratoxin A in grains by immuno-ultrafiltration and HPLC-fluorescence detection after postcolumn derivatisation in an electrochemical cell.

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 µg kg(-1) and 1 µg kg(-1). The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Immuno-ultrafiltration as a new strategy in sample clean-up of aflatoxins.

The present paper describes the development of a new clean-up strategy for the analysis of aflatoxins (AFs) in food. The sample preparation method is based on immuno-ultrafiltration (IUF) which, in contrast to immunoaffinity chromatography, makes use of antibodies in free form. After selecting an appropriate ultrafiltration (UF) device and optimizing different operation conditions the IUF method was applied to the clean-up of maize and rice. Quantification of AFs was carried out by HPLC and fluorescence detection, after postcolumn derivatization in a Kobracell. The IUF method was shown to be as selective as sample clean-up using commercial immunoaffinity columns. Recovery rates and RSD for the AFs G(2), G(1), B(2) and B(1) in spiked rice were found to be 76 +/- 3, 76 +/- 2, 83 +/- 5 and 99 +/- 14%, respectively. The analysis of a FAPAS (food analysis performance assessment scheme) maize material resulted in AFs concentrations which were in the range assigned by the producer of the reference material.