Ablation to Allergen-epitopes of Cow's Milk Proteins by Proteases from Pitcher Fluid of Nepenthes×Mirabilis (Lour.) Druce

Objective: The aim of this study was to abate the allergenicity of the major allergenic proteins in cow's milk by utilizing the proteases in pitcher fluids of Nepenthes mirabilis (Lour.) Druce. Methods: The pitcher fluid of Nepenthes×Miranda was firstly collected, and the protease solution with the proteolytic activity of 126.276 U/mL was obtained by filtration, ultrafiltration with a 5 kDa membrane, vacuum freeze-drying and re-solubilization. Next, these enzymes were used to digest proteins of cow's milk, and the products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then the peptide products were compared with database of allergen-epitopes, so as to determine the cleavage of the allergen-epitopes. Results: The peptide bonds of K, V, F, R, Y, I, S as well as L in the P1 positions and D, A, V, I, L, F, E, R, Y, N, T, Q, S, W as well as P in the P1' positions of the major allergenic proteins in cow's milk were effectively digested, and the cleavage sites were located in the linear epitopes. What's more, both epitopes located on the surface of the proteins and those located in the interior were digested. Specifically, K-V in epitope E_α-LA1 from α-LA and K-V as well as K-A in epitope E_α-LA2 were cleaved at a high frequency. Peptide bonds at epitopes from β-LG which were cleaved at a high frequency were L-D, V-Y, and Y-V in E_β-LG1 and E_β-LG2. The most of R-F and F-F in epitope E_αs1-CN1 from αs1-CN were cleaved at high frequencies. What's more, L-N, R-Y, K-F as well as K-T in epitope E_αs2-CN1 from αs2-CN and K-T, K-L as well as K-I in epitope E_αs2-CN2 were digested with high frequency. And L-Q, S-W, D-V, L-T, T-D as well as E-N in epitopes E_β-CN1 and E_β-CN2 from β-CN were digested with high frequency. Conclusion: The proteases from pitcher fluid exhibited different destructive effects on the allergen-epitopes of cow's milk proteins. Besides, the pitcher liquid of Nepenthes×Miranda is expected to be the resource for the development of novel proteases and the reuse of agricultural wastes will be achieved.