AbstractBACKGROUNDBee venom (apitoxin) is a complex mixture of enzymes, peptides, amines, and other chemicals that presents diverse pharmaceutical activities. Melittin, a small peptide with 2.84 kDa, represents about 50% of this venom, on a dry basis. However, allergenic compounds, such as phospholipase A2 and hyaluronidase (enzymes with a molecular weight of 19 and 38 kDa, respectively), impair the direct use of apitoxin. The membrane separation process can be considered a potential approach for the fractionation of those components. Therefore, the cross‐flow ultrafiltration with a 10 kDa regenerated cellulose membrane was used to separate melittin from apitoxin, leading to lower fouling in the membrane.RESULTSThe strategy was divided into two steps: a 22 factorial design was conducted to evaluate apitoxin concentration and transmembrane pressure, followed by the study of flux decline and fouling mechanisms using the modified Hermia model. The best experimental condition led to 70% of melittin recovery and 99% of rejection of phospholipase A2. Membrane cleaning depicted a recovery of around 92% of flux and reduced 94% of transport resistance. The fouling mechanism revealed three mechanisms acting together in the process.CONCLUSIONThe results show significant values regarding the recovery of melittin and the removal of phospholipase A2, indicating that the cross‐flow ultrafiltration of apitoxin is an attractive option for the isolation of melittin, which has a high market value. © 2020 Society of Chemical Industry (SCI)