The Maillard reaction that occurs during food processing is believed to induce the formation of radical species at high temperatures. This study was carried out to determine whether radical-sensitive Trp is affected during the glycation of β-lactoglobulin (βLG) by lactose in the presence of iron and vitamin C at 115 °C for 6 min. The early glycation, measured as furosine, increased nonlinearly with time, but the formation of fluorescent advanced glycated products (AGEs) was almost exponential, explaining most of the blockage of lysine residues (up to 70%). At the same time, 46−55% of Trp residues were lost, and the Trp concentration was negatively correlated with the fluorescent AGEs. The Trp fluorescence quantum yield decreased considerably due to changes in the protein conformation upon heating with lactose. Trp degradation and AGEs formation were significantly greater in proteic fraction soluble at pH 4.6 than in insoluble fraction. The radicals released during the advanced Maillard step could be responsible for Trp oxidation. Keywords: β-Lactoglobulin; heat denaturation; tryptophan; Maillard reaction

New liquid UHT milks supplemented with iron (0.9-1.4 mg/100 ml), vitamin C (1-7 mg/100 ml), lactose (2-4 g/100 ml) and linoleic acid (200-400 mg/100 ml), named growth milks, have recently become available to satisfy the specific nutritional needs of children aged 1-3 years. But the iron-vitamin C mixture could activate the lactose-induced Maillard reaction and tryptophan (Trp) oxidation in proteins. We have therefore examined the Amadori product and Trp concentrations of these milks. Forty-two commercial growth milks from five firms were analysed for the Maillard reaction and the soluble protein Trp content and compared with 64 UHT milks. The furosine concentration of total proteins was two to four times higher in 'growth' milks than in standard UHT milks, indicating a proportional loss of available lysine. The Trp fluorescence of undenatured proteins soluble at pH 4.6 was almost three times lower in 'growth' than in standard milks and Trp concentration 36% lower suggesting destruction of this oxidation-sensitive amino-acid. The mechanism of Trp destruction remains to be elucidated, and the roles of iron and Amadori products determined.

The in vitro oxidation of tryptophan (Trp) by pro-oxidant systems such as iron-ascorbate indicates that Trp is a target for oxygen radicals in vivo. The Trp in albumin and lipoproteins has been reported to be actively oxidized by hydroxyl radical (HO(•)) generating systems such as copper-ascorbate or PUFA (polyunsaturated fatty acids) respectively. The super-physiological concentrations of the oxidants used in these studies prompted us to examine the effect of low copper and ascorbate concentrations on Trp oxidation. Trp (10-5000 μmol/L) was incubated with 1.5 μmol/L copper plus ascorbate (0.113 and 1.13 mmol/L) at 37°C and its oxidation followed by fluorescence and high-performance liquid chromatography. The percentage of Trp oxidized by the ascorbate-copper system was inversely related to its concentration and positively related to the ascorbate concentration. High concentrations of Trp (above 50 μmol/L for 0.113 mmol/L and 500 μmol/L for 1.13 mmol/L ascorbate) are not significantly oxidized in the presence of ascorbate. The large drop in the percentage Trp oxidation at higher concentrations may be due to the chelation of copper by Trp. High concentrations of Trp (over 50 μmol/L) strongly prevented ascorbate oxidation by copper, and therefore inhibited the production of HO(•) needed for Trp oxidation. Protein Trp is less readily oxidized by the ascorbate-copper system than free Trp. Proteins chelate copper much better than Trp, and so inhibit its oxidative activity, at least against ascorbic acid.