Die Quantifizierung von Aminosäurenisomeren in Lebensmitteln mittels chiraler Gaschromatographie-Massenspektrometrie im Hinblick auf die Relevanz und die Entstehungsmechanismen von D-Aminosäuren

Abstract

Quantification of amino acid isomers in food using chiral gas chromatography - mass spectrometry with regard to relevance and mechanisms of the formation of D-amino acids

1. Subject and aim of the investigation

Different groups of microbial fermented foodstuffs were investigated qualitatively and quantitatively for their amounts of free D- and L-amino acids (AA). Patterns of AA-isomers were determined with regard to the possibility of food characterization by the evaluation of typical chemical marker-AAs, that could serve as indicators for quality and authenticity control of fermented foods.

Further, time and temperature dependent kinetics of AA-racemization were determined in solutions of a standard AA-mixture in aqueous acetic acid (pH 2,5) and in a microbial fermented wine vinegar. The deviating sensitivities of certain AAs towards racemization at moderate pH values in comparison to their respective racemization rates in strong mineral acids should be elucidated Additionally, the influence of the Maillard reaction on the rate of racemization of certain AAs was determined.

The influence of microwave heating of gelatin, and a partial hydrolysate of gelatin, on the formation of the antifibrotic and therefore potentially toxic AA cis-4-Hydroxy-L-proline (cis-4-L-Hyp) was investigated in order to prove a report on the generation of cis-4-L-Hyp due to microwave heating of gelatin from its epimer trans-4-L-Hyp which is a natural component of gelatin.

2. Methods

Liquid samples (vinegar, beer) were treated with DOWEX-cation exchanger to isolate the AAs. Solid samples (grains, malts, hops) were milled and suspended in 0,1 M HCl followed by de-fatting and protein precipitation. Then, the supernatants were subjected to the cation-exchanger.

Gelatin and partial hydrolysates of gelatin were totally hydrolyzed under standard conditions (6 M HCl, 110 °C, 24 h) to release the AAs from the respective proteins and peptides.

Gas chromatographic separation of AA-isomers was carried out using different perfluoroacyl AA alkylester-derivatives on the chiral stationary phase Chirasil-L-Val. For highly sensitive and specific detection a mass spectrometer was chosen. Quantification was performed via the internal standard L-norleucine.

3. Results and conclusions

The chemical markers D-alanine (D-Ala), D-aspartic acid (D-Asp) and D-glutamic acid (D-Glu) characteristic for microbial fermentation were detected in all samples of fermented foods.

Depending on the raw materials used for vinegar production characteristic patterns of AAs were determined. Italian balsamic vinegars (Aceto Balsamico di Modena) showed a significant increase of the absolute and relative amounts of D-Ala and D-proline (D-Pro) during maturation. Especially D-Pro might serve as an indicative for prolonged ripening and ageing. In sherry vinegars and matured red wine vinegars high relative amounts of D-AAs were detected. The absolute amounts, however, were much lower than in balsamic vinegars.

Results of the time and pH-dependent racemization kinetics of proteinogenic AAs, determinable with the GC-MS-method used, showed that Pro does racemize to a negligible extent at moderate acidic pH (pH 2-3). Therefore, an acid catalyzed mechanism for the racemization of Pro in balsamic vinegars could be excluded.

The maximum rate of racemization of Asp at pH 2,5 was explained by the formation of a planar intermediate which is stabilized by an intramolecular hydrogen bond between [beta]-carboxyl group and [alpha]-carboxylate group resulting in a seven membered ring system. For Ser a similar intermediate was postulated via formation of a six membered cycle between the hydrogen atom of the hydroxyl group and the negative charged oxygen atom of the carboxylate group. Therefore, acid catalyzed racemization of Asp and, to a lower extent, Ser might contribute to the amounts of D-AAs detected in acidic fermented foods.

Heating of a mixture of glucose and fructose together with L-Pro led to a very high rate of Pro-racemization in the course of the so-called Maillard-Reaction. This might explain the time-dependent increase of the ageing indicator D-Pro in balsamic vinegars during maturation.

The quantification of AA-enantiomers in beers and raw materials used for manufacturing revealed that raw materials contribute to a negligible (hops) or minor (grains, malts) extent to the D-AA content of beer. Therefore, the amounts of D-AAs detected in beers were mainly attributed to the activity of the racemases of the microorganisms used for brewing. Common top- and bottom-fermented beers like "Altbier", wheat beer, Pilsener or lager contained the typical marker-AAs D-Ala, D-Asp and D-Glu in low amounts, as well as D-Pro in traces. However, in special beers produced by the addition of Saccharomyces-yeast and lactic cultures ("Berliner Weisse") or by spontaneous fermentation using wild yeasts together with lactic cultures (Belgian lambic beers) significantly higher relative and absolute amounts of D-AAs were detected. "Berliner Weisse" beers that underwent bottle conditioning (secondary fermentation) with Lactobacillus delbrueckii contained more than 20% D-Pro. From the data it is concluded that the enantioselective AA-analysis is in particular suitable for the characterization of special beers that are subjected to extraordinary fermentation conditions.

Heating of gelatin and an enzymatic partial hydrolysate of gelatin in a common microwave oven did not, as assumed in the literature, lead to the formation of the antifibrotic and therefore potentially hazardous AA cis-4-L-Hyp. Amounts of ca. 5% cis-4-D-Hyp detected even in untreated controls were shown to be an artifact, resulting from the acidic total hydrolysis of the samples. Therefore, a possible health risk for consumers due to the microwave-induced formation of potentially toxic cis-4-L-Hyp in foodstuffs containing gelatin or partial hydrolysates of gelatin can be excluded.

Zur Frontpage des Volltextes	Zur "Giessener Elektronischen Bibliothek"
Fragen und Kommentare bitte an: koordinierung@ub.uni-giessen.de	Zuletzt geändert am 28.02.2000