Polyphenoloxidase (PPO) and peroxidase (POD) are enzymes that need to be inactivated or inhibited in order to avoid undesirable browning reactions and the loss of sensorial or nutritional quality of fruits and vegetables. As a promising “gentle” alternative to traditional methods, such as pasteurisation or the use of antibrowning compounds, the present study investigates cold plasma's ability to inactivate PPO and POD in a model food system, as well as possible inactivation mechanisms. The study shows that cold plasma is capable of reducing the activity of both PPO and POD in the model food system. The activity of PPO was reduced by about 90% after a treatment time of 180 s. POD was more stable and was reduced by about 85% after 240 s. Circular dichroism and tryptophan fluorescence measurements indicate that the reason for their loss of activity is based on a plasma-induced modification of their secondary structure. A decrease in the alpha-helix content was accompanied by an increase of the percentage of beta-sheet regions. Reduced and red-shifted tryptophan fluorescence intensities supported these changes. Industrial relevance The quality of freshly cut fruits and vegetables greatly depends on the activity of naturally occurring enzymes such as PPO and POD, which catalyse browning reactions at cut surfaces. The presented study shows that cold plasma, as a promising non-thermal pasteurisation technology, is capable of reducing the activity of these enzymes in a model food system. In addition, it describes the impact of different treatment parameters and gives insights into inactivation mechanisms. The results contribute to the understanding of cold plasma effects on enzyme activity and could be a basis for a possible industrial implementation.

Cold plasma effects on enzyme activity in a model food system

Food and beverages contain protein glycation adducts--both early-stage adducts and advanced glycation endproducts. We determined the concentrations of glycation adducts in selected food and beverages by liquid chromatography with triple quadrupole mass spectrometric detection. Cola drink contained low concentrations of glycation free adducts, whereas pasteurised and sterilised milk were rich sources of heat-stable glycation adduct residues--Nepsilon-carboxymethyl-lysine and Nepsilon-carboxyethyl-lysine. Laboratory rodent food was a rich source of advanced glycation endproducts. Measurement of glycation adducts in 24 h urine samples of normal and diabetic rats indicated that < 10% of glycation adduct residue consumption was excreted. Induction of diabetes by streptozotocin led to a 2-fold increase in urinary excretion of Nepsilon-carboxymethyl-lysine and a 27-fold increase in urinary excretion of methylglyoxal-derived hydroimidazolone Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine - the latter was decreased by high-dose thiamine therapy that also prevented the development of nephropathy. We conclude that cola drinks are a poor source of glycation adduct whereas thermally processed milk is rich in glycation adducts. Dietary glycation adducts residues probably have low bioavailability. Experimental diabetes is associated with a marked increase in exposure to endogenous formation of methylglyoxal-derived hydroimidazolone which is linked to the development of diabetic nephropathy.

Assay of advanced glycation endproducts in selected beverages and food by liquid chromatography with tandem mass spectrometric detection.

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly, the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid, sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine, and a sensitive single cell assay to monitor System L amino acid transport. Kynurenine is an immunomodulatory aryl hydrocarbon receptor (AHR) ligand. Here the authors show, using a new flow cytometry based method, that kynurenine needs to be transported across the plasma membrane of activated T cells by the transporter protein SLC7A5 to activate the AHR.

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Single cell analysis of kynurenine and System L amino acid transport in T cells.

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.

Several kynurenine derivatives including N'-formylkynurenine were prepared in high purity by the ozonization of the corresponding indole compounds. The fluorescence characteristics of those derivatives were examined in connection with the use of their fluorophores as reporters for the local environment of tryptophan in proteins. Kynurenine is a weak emitter of fluorescence, with an emission maximum at 480 nm on excitation at 365 nm. With decreasing solvent polarity, the fluorescence intensity increases logarithmically and the emission maximum shifts to blue. A linear relation between these fluorescence characteristics and solvent polarity exists when the polarity is shown in terms of dielectric constant. N'-Formylkynurenine is a somewhat stronger emitter of fluorescence than kynurenine. The emission maximum is 434 nm on excitation at 325 nm and it shifts to blue in solvents of low polarity. This blue shift is also linear with respect to the dielectric constant of the solvent. Other factors influencing kynurenine fluorescence and N'-formylkynurenine fluorescence examined were neighboring groups, ionic strength, temperature, and protein denaturants. Based on the results of the present investigation, the local environment of tryptophan 62 in hen egg-white lysozyme was examined using Kyn 62-lysozyme.

Fluorescence Characteristics of Kynurenine and N′-Formylkynurenine, Their Use as Reporters of the Environment of Tryptophan 62 in Hen Egg-White Lysozyme

Tryptophan was quantitatively oxidized to kynurenine or N′-formylkynurenine on methanolic ozonization and subsequent reduction of the ozonization product, which was identified as N′-methoxyhydroperoxymethylkynurenine by carbon-13 NMR spectroscopy. With dimethyisulfide this peroxidic intermediate was usually reduced to N′-formylkynurenine, but in the acidic condition it was converted to kynurenine.

Yasuhiro Katsuragi

Papers

Fluorescence Characteristics of Kynurenine and N′-Formylkynurenine, Their Use as Reporters of the Environment of Tryptophan 62 in Hen Egg-White Lysozyme

Quantitative ozone-oxidation of tryptophan to n′-formylkynurenine and kynurenine

Quantitative ozone-oxidation of tryptophan to n′-formylkynurenine and kynurenine