Milk protein modulates antioxidant activity and metabolome stability in coffee beverages during thermal processing

Milk is frequently added to coffee to enhance its flavor; however, its effect on antioxidant compounds and related metabolite profiles remains poorly characterized. In this study, the functional properties and free metabolites of the milk-infused coffee were analyzed under thermal conditions. Results indicated that milk addition facilitated the formation of polyphenol-protein complexes, increasing the antioxidant properties. For instance, when milk was added to achieve a protein concentration of 90 mg/mL, the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging capacity of the coffee-milk system increased from 33.55% to 49.37%. Furthermore, high coffee concentration tended to increase the average particle size of the coffee-milk beverage, which made the overall system unstable, evidenced by a higher polydispersity index. An increase in milk protein concentration was observed to result in an enhanced foam expansion rate, accompanied by a slight reduction in foam stability. Meanwhile, the results of the sensory evaluation showed that the increase in milk protein concentration changed the objective sensory attributes of the coffee-milk beverages including bitterness, astringency, and sweetness, and improved the overall acceptance. When the milk protein concentration reached 90 mg/mL, the overall sensory acceptance score of coffee-milk beverages increased to 7.40 out of 10, which was significantly higher than the 3.88 score for plain black coffee. Comparative analysis by native and non-native electrophoresis revealed that there are simultaneous noncovalent and covalent interactions between the active ingredient and milk proteins. High-performance liquid chromatography quadrupole TOF MS/MS demonstrated that the addition of milk resulted in a significant change on the metabolite profile in the coffee-milk beverage in terms of both type and content, such as dicaffeoylquinic acid and feruloyl-quinolactone, which was independent of the temperature at which the coffee-milk beverage was prepared. The central factor in the binding of molecules in coffee is milk protein, which may contribute to changes in the physiochemical and nutritional properties of coffee. It is thus possible to develop milk coffee with an enhanced overall quality and optimized biofunctional properties by examining the composition ratio and processing conditions. In conclusion, milk protein acts as a key mediator in coffee molecule binding, modifying antioxidant activity and metabolite profiles. This mechanism lays the foundation for the targeted design of milk-coffee formulations with optimized biofunctional properties through precise control of composition ratios and processing parameters.