Effect of pyridoxamine on the glycation via maillard reaction of bovine β-lactoglobulin with galactose

Abstract

Over the past few years there has been an increased interest in deliberately promoted Maillard reaction (MR) to obtain glycoconjugates with improved functionalities in relation to the initial proteins. In this respect, depending on the functional property, it is of paramount importance to carry out an exhaustive control of the progress of the reaction. For this, besides controlling reaction conditions such as temperature, time, water activity and pH, the use of inhibitors of the advanced glycation such as pyridoxamine (PM), a natural intermediate of vitamin B6 metabolism, may allow a more thorough control of MR. Thus, PM has been demonstrated to be an adequate in vitro and in vivo inhibitor of transformation of the Amadori compounds to AGEs (advanced glycation end-products) acting on several pathways of MR. However, whilst all these studies were carried out in solution simulating in vivo physiological conditions using aldoses, scarce data have been reported on the usefulness of PM as an inhibitor of MR in food systems and its impact on the protein aggregation. Thus, the aim of this work has been the study of the impact of PM on deliberately promoted MR during the glycation of β-lactoglobulin (β-Lg) with galactose (Gal). To favour the formation of covalent complexes, the reaction was done in dry-solid state (0.44 aw), pH 7.0 (0.1 M sodium phosphate buffer), and temperature set at 40 and 50ºC. To investigate the possible interaction between PM and Gal, glycoconjugates stored with and without PM were analyzed by MALDI-TOF-MS and RP-HPLC-UV. Moreover, changes in reducing carbohydrates concentration by the anthrone-sulfuric acid method using 96-well microtitration plates as well as formation of Amadori or Heyns compounds by ion-pair RP-HPLC were assessed. Finally, the effect of PM on the advanced and final stages of MR was evaluated through the aggregation degree and browning determination by SEC and absorbance at 420 nm, respectively. The results showed the formation of interaction products between PM and Gal either in presence or in absence of β-Lg, indicating that PM competes with the free amino groups of β-Lg for the carbonyl group of carbohydrate. Thus, a small inhibitory effect of PM on the initial stages of the MR was pointed out. Furthermore, much lower aggregation and colour formation were observed in the conjugates β-Lg:Gal with PM than without this compound, demonstrating its potent inhibitory effect on the advanced and final stages of the MR. These findings could reveal the usefulness of food-grade inhibitors of the advanced stages of the MR, such as PM, in combination with mild storage conditions for the formation of safer neoglycoconjugates with specific functionalities and without impairing their nutritional quality.