Impact of thermal storage on lysine content and Maillard reaction products in samples of enteral nutrition diets

Abstract

Furosine, carboxymethyllysine (CML), and pyrraline are chemical markers formed at different stages of the Maillard reaction, a non-enzymatic process between reducing sugars and amino groups that occurs during heat treatment. Lysine, an essential amino acid, is particularly susceptible to this reaction and serves as an indicator of nutritional loss in processed foods. This study aimed to evaluate the formation of Maillard reaction products in commercial enteral formulas before and after storage at 50 °C, and to relate these changes to the composition of each formulation. Fourteen enteral formulas from different manufacturers were stored in a forced-air oven at 50 °C for up to 21 days to simulate accelerated shelf-life degradation. The analyzed markers included furosine, carboxymethyl lysine (CML), pyrraline, and available lysine. Furosine and lysine were determined using ion exchange chromatography with UV detection and post-column derivatization with ninhydrin, following acid hydrolysis. Pyrraline was quantified after enzymatic hydrolysis using reverse-phase HPLC. CML was determined after reduction and acid hydrolysis using liquid chromatography coupled with mass spectrometry (LC-MS/MS) in Multiple Reaction Monitoring (MRM) mode. Before storage, furosine levels ranged from 25.8 to 132.5 mg/100 g, with the highest values found in formulas containing maltodextrin, glucose syrup, and concentrated or hydrolyzed proteins — ingredients rich in carbonyl and amino groups that favor early Maillard reactions. After storage at 50 °C, most samples showed a marked increase in furosine, especially sample 3A. Available lysine ranged from 1.0 to 1.9 mg/100 g, with slight reductions after storage; samples 10C and 11C retained higher levels, indicating greater nutritional stability. CML increased significantly in selected samples after storage, reaching over 21 mg/100g. Pyrraline content also increased, particularly in samples which contained glucose syrup, isomaltose, and milk protein concentrates. Studying these markers is essential because they reflect chemical transformations that directly affect the nutritional quality of enteral formulas. Furosine and CML indicate thermal damage and reduced bioavailability of essential amino acids such as lysine, while pyrraline is a representative advanced glycation end-product (AGE) that may have pro-inflammatory or metabolic effects. Monitoring these compounds allows for better understanding of product stability during storage and supports the development of safer, more stable, and nutritionally effective formulas — especially for clinically vulnerable populations who rely on enteral nutrition as their sole dietary source.