Our aim was to entrap lactoferrin (LF) in complex nano-particles of whey protein isolate (WPI)-high methoxyl pectin (HMP) with the ratios of 2:1, 1:1, and 1:2 through acidification at pH values of 3, 3.5, and 4. The zeta-potential, size, sedimentable-complex yield, LF content, encapsulation efficiency, SEM, AFM, FTIR, and DSC of nano-particles were investigated. Our results revealed that almost all analyzed parameters of the final nano-particles were related to preparation pH value, WPI/HMP ratios, and acidification methods In both methods of pre- and post-acidification, the zeta potential was decreased via decreasing of pH from 4 to 3 and particle size was increased at higher HMP ratios to WPI. In general, the pre-blending acidification provided a larger mass of complexes compared with post-blending counterparts. Also, the nano-particles produced by WPI/HMP with the ratio of 2:1 at pH = 3.5 had the smallest sizes. The highest LF content of the complexes as well as the optimal entrapment efficiency was observed at pH = 3.5, in both methods of post and pre-blending. Finally, the pre-blending by a ratio of 2:1 for WPI/HMP was chosen as the optimal treatment for producing nano-particles containing LF. This was confirmed by SEM, AFM, FTIR, and DSC studies.

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