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Establishment and evaluation of different methodologies (specific analysis, metabolic profiling, non-destructive technique) for the analytical comparison of parental and transgenic rice

The objective was to compare the chemical compositions of transgenic and parental rice grains used in the feeding studies.

Two different methodologies were evaluated as tools for an analytical comparison:
- Targeted analysis of (anti-) nutrients and toxicants, and
- Non-targeted analysis of low molecular weight compounds by means of metabolite profiling.

- Targeted analysis
Comprehensive analytical characterization of rice material was performed by application of validated standard protocols. Analysis of the chemical compositions included proximate analysis (water, ash, fibre, starch, sugars, fat, protein), analysis of lipids (fatty acid distribution, phospholipids), amino acids, vitamins (B1, B6, niacin, pantothenic acid, folic acid) and minerals (sodium, potassium, magnesium, calcium, manganese, iron, copper, zinc, molybdenum, phosphorous, selenium). Differences between the lines were assessed for statistical significance and evaluated in the light of natural variations described in literature. In order to check the microbiological quality, fungal and bacterial counts were determined and the material was tested for mycotoxins. As regards contaminants, the material was analysed for heavy metals and pesticides.

Conventional methods for the targeted analysis of minor food constituents usually require laborious sample preparation steps in order to isolate analytes from the food matrix prior to analysis. On-line coupled liquid chromatography–gas chromatography (on-line LC-GC) was evaluated as a technique for the rapid analysis of minor grain constituents preventing time-consuming sample preparation steps. A method based on on-line LC-GC was developed for the analysis of phytosteryl ferulates, a group of minor rice constituents exhibiting antioxidative and cholesterol-lowering activities. On-line LC-GC proved to be a powerful analytical tool for the targeted analysis of minor rice constituents without laborious sample preparation steps.

The suitability of Near Infrared Spectroscopy (NIRS) for non-destructive determination of minor compounds was investigated by using phytic acid, an antinutritional rice grain constituent, as example. Applicability of the methodology for rapid screening of rice samples from different origins regarding low, medium, and high phytic acid contents was demonstrated. Results obtained by NIRS for GM and parental rice lines were in agreement with those determined by the traditional method. Sample preparation is reduced to a minimum by the non-destructive approach.

- Non-targeted analysis
A gas chromatographic metabolite profiling method for unbiased screening of a broad spectrum of compounds was elaborated using rice as model crop. It is based on a fractionation approach which eventually allows non-targeted investigation of major and minor rice grain constituents. General applicability of the technique for analysis of cereals with metabolite compositions similar to rice was demonstrated. Reliability of the profiling approach was proven by investigation of rice, maize and barley samples with known and expected differences on the metabolite level.

Software tools for comparative analysis of metabolite profiling data were developed. They include methods for manual and automated correction of retention times and responses by means of corresponding standards. This allows transfer of normalised datasets into databases and eventually automated unbiased chromatogram comparison. Application of the metabolite profiling methodology to the analysis of GM and conventional rice revealed the suitability of a non-targeted approach for detection of statistically significant differences between metabolite levels in different genotypes.

The usefulness of databases established through investigation of a broad spectrum of conventional rice cultivars for evaluation of differences in metabolite profiling data was demonstrated. The unbiased screening of a broad spectrum of compounds combined with powerful methods for data analysis may help to increase the chances to detect unintended effects due to the application of recombinant DNA techniques.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Informations connexes

Reported by

Technische Universität München
Am Forum 2
85350 Freising-Weihenstephan
Germany
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