Study On The Interaction And Mechanism Between Bovine Whey Protein And Acrylamide In Coffee

Green coffee beans undergo a series of reactions such as Maillard reaction and caramelization during the high-temperature roasting stage,giving coffee a unique flavor and making it one of the high-contribution foods of acrylamide.Acrylamide is a recognized neurotoxin and quasi carcinogen.But there is no evidence that drinking coffee rich in acrylamide is associated with human cancer.The reason for the conflict may be due to dietary habits: coffee products often contain milk in different proportions and forms to reconcile coffee flavor and increase nutrition.This topic proposes the hypothesis of “protein-ligand interaction”,that is,bovine whey protein has a structural site that specifically binds small molecule ligands,and will bind to acrylamide to affect its existence and concentration,and then affect its immunotoxicity.The subject took milk and coffee as the research objects,and focused on the research idea of “molecular interaction-cellular immunotoxicity-molecular structure characterization”.We studied the effect of bovine whey protein on acrylamide concentration through liquid quality detection,and then studied the effect of bovine whey protein on acrylamide immunocytotoxicity through cell experiment.Finally,the molecular state of bovine whey protein combined with acrylamide was studied by molecular docking and molecular dynamics simulation.The research results showed that 678.3 ± 30.0 μg/kg acrylamide was produced after roasting Indonesian robusta green coffee beans at 160 ℃ for 9 min.The concentration of acrylamide in the coffee solution was significantly reduced by 95 %,after adding milk.Five different components of milk with basically the same protein content can reduce the concentration of acrylamide aqueous solution to 40.7 ± 2.3 %,indicating that water-soluble bovine whey protein in milk plays a major role in reducing the concentration of acrylamide.The four bovine whey proteins can reduce the concentration of acrylamide exponentially,among which α-lactalbumin and β-lactoglobulin play a major role.11.92 μmol/L α-lactalbumin or 43.50 μmol/L β-lactoglobulin can reduce the concentration of acrylamide to 20%,and all 13 kinds of amino acids could interact with small molecule acrylamide.Toxicity experiments on mouse RAW264.7 macrophages confirmed that acrylamide would be toxic to macrophages.Increasing the treatment concentration of acrylamide or prolonging the treatment time will lead to the lower cell viability.The median lethal concentration of cells was 70 mmol/L at 8 h exposure.The infected macrophages have poor morphology,weakened adherence ability,and a large number of cells are eliminated,resulting in a significant reduction in the ability of macrophages to phagocytose neutral red and the production of reactive oxygen species(ROS).Both β-lactoglobulin and α-lactalbumin mixed with acrylamide in serum-free medium,can effectively reduce their toxicity to macrophages,restore the cell shape to its original state,and restore the detected values to normal levels or even higher.The recovery effect of α-lactalbumin is better than that of β-lactoglobulin.50 μmol/Lβ-lactoglobulin or α-lactalbumin was added to serum-free medium which contained 70mmol/L acrylamide can increase the survival rate of macrophages to 96.73 % and103.69 % respectively,or increase phagocytosis of neutral red to 107.63 % and 115.00 %respectively.60 μmol/L β-lactoglobulin or α-lactalbumin was added to serum-free medium which contained 80 mmol/L acrylamide can increase the damage of macrophage ROS production to 99.60 % and 115.13 % respectively.The results of molecular docking and molecular dynamics simulation of acrylamide and two bovine whey proteins show that small molecule acrylamide can bind to the hydrophobic groups and polar groups of amino acid residues on β-lactoglobulin or α-lactalbumin,thereby binding within the cavity of the two proteins.The optimal docking model of β-lactoglobulin-acrylamide has a lower ΔG° than α-lactalbumin-acrylamide,but α-lactalbumin can bind acrylamide molecules in more stable ways.The RMSD,RMSF and Rg of the two bovine whey proteins fluctuated slightly before and after combining with acrylamide,but the overall changes were not uniform,indicating that the two bovine whey proteins combined with acrylamide complexes stably.Among them,the structure of α-lactalbumin-acrylamide complex is tighter and more stable.All these results suggest that whey protein in milk can interact with acrylamide in coffee to form a more stable complex structure,thereby reducing the concentration of acrylamide in it,and further reducing the toxicity of acrylamide to macrophages.The ability of α-lactalbumin is better.This paper clarifies the molecular interaction mechanism between different chemical components in milk and coffee,reveals the impact of dietary habits on human health,and provides a theoretical basis for the safety evaluation of“coffee + milk” drinking habits and the development of coffee products.