Preparation of Hypoallergenic Whey Protein Hydrolysate by a Mixture of Alcalase and Prozyme and Evaluation of its Digestibility and Immunoregulatory Properties

Jiyeon Yang1,2,, Se Kyung Lee1,, Young Suk Kim3, Hyung Joo Suh1,4, Yejin Ahn1,*
Author Information & Copyright
1Department of Integrated Biomedical and Life Science, Graduate School, Korea University, Seoul 02841, Korea.
2Maeil Innovation Center, Maeil Dairies Co., Ltd., Pyeongtaek 17714, Korea.
3Department of Food and Nutrition, Ansan University, Ansan 15328, Korea.
4Transdisciplinary Major in Learning Health Systems, Department of Healthcare Sciences, Graduate School, Korea University, Seoul 02841, Korea.

† These authors contributed equally to this work.

*Corresponding Author: Yejin Ahn. E-mail:

© Copyright 2023 Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: May 03, 2023 ; Revised: May 15, 2023 ; Accepted: May 16, 2023

Published Online: May 23, 2023


Whey protein (WP) has nutritional value, but the presence of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) cause allergic reactions. In this study, hypoallergenic whey protein hydrolyate (HWPH) was prepared by decomposing β-LG and α-LA of WP using exo- and endo-type proteases. The enzyme mixing ratio and reaction conditions were optimized using response surface methodology (RSM). Degradation of α-LA and β-LG was confirmed through gel electrophoresis, and digestion, and absorption rate, and immunostimulatory response were measured using in vitro and in vivo systems. Through RSM analysis, the optimal hydrolysis conditions for degradation of α-LA and β-LG included a 1:1 mixture of Alcalase and Prozyme reacted for 10 h at a 1.0% enzyme concentration relative to substrate. The molecular weight of HWPH was <5 kDa, and leucine was the prominent free amino acid. Both in vitro and in vivo tests showed that digestibility and intestinal permeability were higher in HWPH than in WP. In BALB/c mice, as compared to WP, HWPH reduced allergic reactions by inducing elevated Type 1/Type 2 helper T cell ratio in the blood, splenocytes, and small intestine. Thus, HWPH may be utilized in a variety of low allergenicity products intended for infants, adults, and the elderly.

Keywords: hydrolysate; whey protein; response surface methodology; hypoallergenic; infant nutrition

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