Preparation of Whey Protein Hydrolysates Using a Single- and Two-Stage Enzymatic Membrane Reactor and Their Immunological and Antioxidant Properties: Characterization by Multivariate Data Analysis

An initial 5% (w/v), followed thereafter with replacement aliquots of 3% (w/v), whey protein isolate (WPI) (ca. 86.98% Kjeldahl N x 6.38), was hydrolyzed using Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) in an enzymatic membrane reactor (EMR) fitted with either a 10 or 3 kDa nominal molecular weight cutoff (NMWCO) tangential flow filter (TFF) membrane. The hydrolysates were desalted by adsorption onto a styrene-based macroporous adsorption resin (MAR) and washed with deionized water to remove the alkali, and the peptides were desorbed with 25, 50, and 95% (v/v) ethyl alcohol. The desalted hydrolysates were analyzed for antibody binding, free radical scavenging, and molecular mass analysis as well as total and free amino acids (FAA). For the first time a quantity called IC50, the concentration of peptides causing 50% inhibition of the available antibody, is introduced to quantify inhibition enzyme-linked immunosorbent assay (ELISA) properties. Principal component analysis (PCA) was used for data reduction. The hydrolysate molecular mass provided the most prominent influence (PC1 = 57.35%), followed by inhibition ELISA (PC2 = 18.90%) and the antioxidant properties (PC3 = 10.43%). Ash was significantly reduced in the desalted fractions; the protein adsorption recoveries were high, whereas desorption with alcohol was prominently influenced by the hydrophobic/ hydrophilic amino acid balance. After hydrolysis, some hydrolysates showed increased ELISA reactivity compared with the native WPI.