Potential of Subcritical Water Hydrolysis to Valorize Low-Valued Ray-Finned Fish (Labeobarbus nedgia): Effects of Hydrolysis Temperature and Pressurization Agent.

Subcritical water (SCW) hydrolysis was applied to valorize the low-valued ray-finned fish (Labeobarbus nedgia) into valuable protein hydrolysates, employing N2 and CO2 as pressurization agents at varying temperatures (140, 160, 180, and 200 °C). The degree of hydrolysis (DH) and total free amino acid content increased with temperature for both pressurizing agents. The highest DH (54.5 ± 0.4%) and total free amino acid content (210 ± 1 mg/gprot) were observed at 200 °C when CO2 gas was used as the pressurizing agent. Predominantly, glycine and alanine were released for both pressurizing agents. The antioxidant activity, evaluated through three different assays, increased with temperature and was found to be the highest at 200 °C. This study illustrated the advantages of the intensified SCW technology by using CO2 as a pressurization agent in valorizing low-valued ray-finned fish (Labeobarbus nedgia), as animal residue rich in proteins, for the production of valuable protein hydrolysates with a high fraction of valuable free amino acids, which could offer potential applications as a functional ingredient in the food industry.

Whey protein hydrolysates and infant formulas: Effects on physicochemical and biological properties.

Whey protein hydrolysates are recognized for their substantial functional and biological properties. Their high digestibility and amino acid composition make them a valuable ingredient to hydrolyzed whey infant formulas, enhancing both product functionality and nutritional values for infant growth. It is important to understand the functional and biological properties of whey protein hydrolysates for their applications in infant formula systems. This review explored preparation methods of whey protein hydrolysates for infant formula-based applications. The effects of whey protein hydrolysate on the physicochemical and biological properties of hydrolyzed whey infant formulas were summarized. The influences of whey protein hydrolysates on the functional and nutritional properties of formulas from manufacturing to infant consumption were discussed. Whey protein hydrolysates are crucial components in the preparation of infant formula, tailored to meet the functional and nutritional demands of the product. The selection of enzyme types and hydrolysis parameters is decisive for obtaining "optimal" whey protein hydrolysates that match the intended characteristics. "Optimal" whey protein hydrolysates offer diverse functionalities, including solubility, emulsification and production stability to hydrolyzed whey infant formulas during manufacturing processes and formulations. They simultaneously promote protein digestibility, infant growth and other potential health benefits, including reduced allergenic potential, as supported by in vitro, in vivo and clinical trials. Overall, the precise selection of enzymes and hydrolysis parameters in the production of whey protein hydrolysates is crucial in achieving the desired characteristics and functional benefits for hydrolyzed whey infant formulas, making them critical in the development of infant nutrition products.

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of ß-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Production of Bioactive Peptides from Hake By-Catches: Optimization and Scale-Up of Enzymatic Hydrolysis Process.

Fish by-catches, along with other fish side-streams, were previously used as raw material for the production of fishmeal and fish oil but appropriate handling allows their use in more valuable options. The aim of this research was to valorize undersized hake (Merluccius merluccius) as a model of using fish by-catch from the Bay of Biscay to produce protein hydrolysates with bioactivities. Six enzymes, with different proteolytic activities (endo- or exoproteases) and specificities, were tested to produce protein hydrolysates. Products obtained with an endoprotease of serine resulted in the most promising results in terms of protein extraction yield (68%), with an average molecular weight of 2.5 kDa, and bioactivity yield (antioxidant activity = 88.5 mg TE antioxidant capacity/g fish protein; antihypertensive activity = 47% inhibition at 1 mg/mL). Then, process conditions for the use of this enzyme to produce bioactive products were optimized using Box-Behnken design. The most favorable process conditions (time = 2 h, solids = 50% and enzyme/substrate = 2% with respect to protein) were scaled up (from 0.5 L to 150 L reactor) to confirm laboratory scale and model forecasts. The results obtained in the pilot-scale testing matched the outcomes predicted by the model, confirming the technical viability of the proposed process.

Production and Characterization of Heme Iron Polypeptide from the Blood of Skipjack Tuna (Katsuwonus pelamis) Using Enzymatic Hydrolysis for Food Supplement Application.

Organic heme iron in the form of heme iron polypeptide (HIP) is a bioavailable form of iron that can be used for dietary supplements. However, one practical challenge with HIP is that the quality of HIP prepared with different batches of raw material could lead to HIP products with inconsistent characteristics. In this study, skipjack tuna blood, a by-product in canned tuna industry, was converted to HIP at different degrees of enzymatic hydrolysis. The variation in HIP physical-chemical characteristics from different batches was evaluated, including composition, solubility, and molecular weight distribution. It was found that the batch variation had no effect on HIP composition and solubility; however, the degree of hydrolysis (DH) and the size of peptides that interact with heme greatly influenced HIP solubility at pH 2. Tuna-HIP with a low DH (DH, 8%) had 1.76-fold greater solubility than tuna-HIP with a high DH (DH, 32%). High-performance liquid chromatography (HPLC) revealed that tuna-HIP with a low DH had a molecular weight ranging from 1 kDa to 5 kDa. In summary, HIP-derived tuna blood was found to contain 70.54 ± 3.22 mg/100 g of iron and exhibit good solubility at 58.0 ± 2.16% at pH 2. Thus, tuna-HIP with a low DH might be a suitable functional ingredient for iron fortification of food.

Degree of Hydrolysis Regulated by Enzyme Mediation of Wheat Gluten Fibrillation: Structural Characterization and Analysis of the Mechanism of Action.

The impact of different degrees of hydrolysis (DHs) on fibrillation when trypsin mediates wheat gluten (WG) fibrillation has not been thoroughly investigated. This study discussed the differences in amyloid fibrils (AFs) formed from wheat gluten peptides (WGPs) at various DH values. The results from Thioflavin T (ThT) fluorescence analysis indicated that WGPs with DH6 were able to form the most AFs. Changes in Fourier Transform Infrared (FTIR) absorption spectra and secondary structure also suggested a higher degree of fibrillation in DH6 WGPs. Analysis of surface hydrophobicity and ζ-potential showed that DH6 AFs had the highest surface hydrophobicity and the most stable water solutions. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) images revealed the best overall morphology of DH6 AFs. These findings can offer valuable insights into the development of a standardized method for preparing wheat gluten amyloid fibrils.

Intervention Effects of Deer-Tendon Collagen Hydrolysates on Osteoporosis In Vitro and In Vivo.

Deer tendon, a deer processing byproduct, is an excellent protein source for the preparation of peptides for improving osteoporosis by its high protein content and high nutritional value. The optimal process of collagen acid extraction was implemented and the results showed that the acid concentration was 7%, the material-liquid ratio was 1:25, and the soaking time was 48 h. DTCHs could promote MC3T3-E1 cell proliferation and increase alkaline phosphatase activities in vitro. In addition, compared with the model group, the DTCHs treatment groups with an oral dosage of 350, 750, and 1500 mg/kg rat/day could significantly improve the shape, weight, bone mechanics, and alkaline phosphatase activities of tail-suspended mice. Bone microstructure and mineralization also recovered significantly in vivo. This result is expected to provide the structural and biological information for DTCHs-based functional foods.

Protein Quality and Protein Digestibility of Vegetable Creams Reformulated with Microalgae Inclusion.

Microalgae are considered a valuable source of proteins that are used to enhance the nutritional value of foods. In this study, a standard vegetable cream recipe was reformulated through the addition of single-cell ingredients from Arthrospira platensis (spirulina), Chlorella vulgaris, Tetraselmis chui, or Nannochloropsis oceanica at two levels of addition (1.5% and 3.0%). The impact of microalgae species and an addition level on the amino acid profile and protein in vitro digestibility of the vegetable creams was investigated. The addition of microalgae to vegetable creams improved the protein content and the amino acid nutritional profile of vegetable creams, whereas no significant differences were observed in protein digestibility, regardless of the species and level of addition, indicating a similar degree of protein digestibility in microalgae species despite differences in their protein content and amino acid profile. This study indicates that the incorporation of microalgae is a feasible strategy to increase the protein content and nutritional quality of foods.

Comparative efficiency of native and non-native starter culture in the production of bio-silage using composite waste from fish and vegetables.

The efficiency of native and non-native starter cultures in the production of bio-silage using composite waste from fish and vegetables was studied. An ensilage experiment was conducted in a natural way (without starter culture) of composite waste (fish to vegetable at 80 to 20%) to isolate the native fermentative microflora. An Enterococcus faecalis strain isolated from the natural ensilage of composite waste showed higher efficiency over other commercial LAB strains generally used for ensilation. A total of 60 isolates were screened and characterized biochemically from ensilaged composite waste. Among them, 12 proteolytic and lipolytic positive isolates were identified as Enterococcus faecalis, based on a BLAST search of the 16S rRNA gene sequences. Subsequently, composite bio-silage was prepared by inoculating starter cultures with three (3) treatments T1 (native-Enterococcus faecalis), T2 (non-native-Lactobacillus acidophilus), T3 (a mixture of E. faecalis and L. acidophilus) and compared with control (composite bio-silage without starter culture). The highest non-protein nitrogen (0.78 ± 0.01 mg of N /100 g) and degree of hydrolysis (70.00 ± 0.06% of protein/100 g) was seen in the T3 sample, and the lowest (0.67 ± 0.02 mg of N/100 g and 50.40 ± 0.04% of protein/100 g) was seen in the control. At the end of ensilation, the pH fell (5.95-3.88) in conjunction with the formation of lactic acid (0.23-2.05 g of lactic acid/100 g), and the lactic acid bacteria count nearly doubled (log 5.60-10.60). The lipid peroxidation products PV (0.11-0.41 milli equivalent of oxygen/kg of fat) and TBARs (1.64-6.95 mg of malonaldehyde/kg of silage) were changed within a reasonable range in the following pattern Control > T2 > T3 > T1, which led to oxidatively stable products. The findings revealed that native starter culture E. faecalis, which can be employed as a single or in combination with non-native L. acidophilus, performed better in the bio-ensilation process. Additionally, the finished composite bio-silage can be used as a novel, protein-carbohydrate rich feed component to help manage wastes from both sectors.

Feasibility of using a realistic food bolus for semi-dynamic in vitro gastric digestion of hard cheese with pH-stat monitoring of protein hydrolysis.

Oral processing of solid foods leads to boluses made of a human saliva and particles distributed in the size range âˆ¼ 0 to 5 mm. However, studies on the release of nutrients from realistic solid food boluses during digestion are scarce because such mechanisms are difficult to investigate in vivo, and in vitro experiments generally recommend to extensively mince solid foods during the oral stage. Similarly, it has previously been shown that the peptic hydrolysis of protein solutions during in vitro gastric digestion can be monitored by acid titration in both static and dynamic pH conditions, but such approach has never been evaluated in the presence of particles of several millimetres in size. The first objective of the study was therefore to test the feasibility of using a realistic food bolus for gastric digestion studies with a pH-stat monitoring of proteolysis, using Emmental cheese as a solid food and with consideration of gastric acidifying kinetics. Degree of hydrolysis (DH) of proteins was monitored from two series of experiments performed in the presence and absence of pepsin. Other DH measurements, estimated from an independent approach based on the amount of free NH2 groups (OPA method) contained by peptides released in the supernatant (UV absorbance) validated the pH-stat results. A second objective of this work was to test the possible influence of human saliva on gastric proteolysis (in comparison with a water-based bolus). Results showed that saliva slightly delayed initiation of proteolysis, which could be explained by the slightly higher initial pH of the saliva-based bolus, but had no statistical effects on pepsin activity. We conclude that acid titration with a pH-stat system can be a valuable approach to monitor the gastric in vitro proteolysis of realistic solid food boluses in dynamic pH conditions.

Investigation of the optimal production conditions for egg white hydrolysates and physicochemical characteristics.

This study aimed to investigate the potential of egg white protein hydrolysate (EWH) as a functional food by identifying the optimum production conditions for EWH with response surface methodology (the results of the sensory evaluation were considered as an essential quality indicator). At the same time, its physicochemical and biological activity was also evaluated. The optimal economic production conditions were selected: substrate concentration of 12.5%, enzyme content of 7.5%, and hydrolysis time at 100 min. The degree of hydrolysis (DH %) was 13.51%. In addition, to the better acceptance of the evaluation, it also helps to reduce the production cost of the protein hydrolysate, which is beneficial to future processing and applications. The antioxidant capacity experiments showed that EWH has good antioxidant activity, which presents a dose-dependent relationship. Hence, this study provides a theoretical basis for future research and application of EWH for processing applications, including dietary supplementation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05708-0.

Gastric devolution of transglutaminase-induced acid and rennet-induced casein gels using dynamic DIDGI® and static COST action INFOGEST protocols.

Limited studies in the literature have compared in vitro dynamic and in vitro static protocols for modelling the gastric digestive process of food systems. This experiment explores the differences between two different in vitro approaches to the devolution of a transglutaminase-induced acid gel (TG, pH 5.1-5.3) and rennet-induced gel (RG, pH 6.5-6.7). Gels were exposed to a simulated oral phase, followed by either the dynamic DIDGI® or static COST action INFOGEST protocol to simulate gastric conditions. Protein hydrolysis was evident from 15 min onwards for TG exposed to the dynamic protocol where levels continued to increase at a steady rate. In contrast, RG exhibited a notable lag-phase before levels increased from around 60 min onwards. Under the static protocol, protein hydrolysis was observed for both TG and RG upon exposure to the gastric environment which continued to increase over time. Despite these differences, similar levels of protein hydrolysis were found for TG and RG at the gastric endpoint using either protocol demonstrating that both the dynamic DIDGI® and static COST action INFOGEST methods provide a suitable and comparable environment for the in vitro digestion of casein protein under simulated gastric conditions.

The Degree of Hydrolysis and Peptide Profile Affect the Anti-Fatigue Activities of Whey Protein Hydrolysates in Promoting Energy Metabolism in Exercise Mice.

The purpose of this study was to investigate the effects of characteristics of whey protein hydrolysates (WPHs) on energy metabolism in exercise mice. Results showed that high-degree of hydrolysis (DH) hydrolysates (22%, H-Alc and H-AXH) showed better anti-fatigue effects than low-DH hydrolysates (10%, L-Alc and L-AXH) in enhancing energy substances and reducing metabolic byproducts. It might be related to the higher content of components less than 3 kDa in H-Alc and H-AXH (92.35 and 81.05%, respectively) and higher intensities of small peptides containing two to nine residues. Moreover, Western blot results revealed that WPHs maintained the energy balance in exercise mice by regulating the AMP-activated protein kinase (AMPK) and mTOR signaling pathways. Notably, H-Alc had higher intensities of peptides containing two to five residues than H-AXH and these peptides were rich in essential amino acids, which might explain why H-Alc exhibited better effects in decreasing protein metabolites. Meanwhile, H-AXH contained more free amino acids, especially Leu, which might contribute to its ability to promote glucose consumption in muscle. Furthermore, 40 peptides with two to nine residues and high intensities (>5 × 105) were screened from H-Alc and H-AXH and predicted by bioinformatics tools. Among them, LLL, LLF, GTW, AGTW, and ALPM showed high bioavailability, cell permeability, and potential bioactivity.

Effect of multi-mode sweep frequency ultrasound pretreatment on properties of the zeins and ACE inhibitory peptides activity of the hydrolysates.

Effects of sweep frequency ultrasound (SFU) pretreatment of a new multifunctional ultrasonic equipment on hydrolysis characteristics of zeins and angiotensin-converting enzyme (ACE) inhibitory activity of zein hydrolysates were investigated. Degree of hydrolysis of zeins reached the highest of 25.93 % and 25.72 % at 40 kHz and 25/40 kHz, respectively. While 25/40 kHz increased solubility, surface hydrophobicity, particle size uniform of zeins and ACE inhibitory activities of the hydrolysates significantly. Endogenous fluorescence indicated that 25/40 kHz promoted unfolding of protein molecules and exposure of hydrophobic residues, thereby facilitating enzymatic hydrolysis. Circular dichroism spectrum and Fourier transform infrared spectrometer illustrated that 25/40 kHz unfolded protein molecules and decreased α-helical contents remarkably. Gel permeation chromatography showed that more small-molecule active peptides were obtained from hydrolysates at 25/40 kHz. In conclusion, SFU pretreatment at 25/40 kHz with the new equipment before proteolysis is an efficient method to improve ACE inhibitory activity of the hydrolysates.

A basis for IgY-themed functional foods: Digestion profile of oral yolk immunoglobulin (IgY) by INFOGEST static digestion model.

Although yolk immunoglobulin (IgY) has been reported its functions of antiviral, antibacterial, energy balancing, and gut microbiota regulating, its gastrointestinal digestion process and the digestion products have not been studied, which is of great significance for the development of IgY-themed functional foods. This work investigated the digestion behaviors of oral IgY by static digestion simulation in vitro. IgY showed low digestibility (23.97%) in the gastric phase but was highly digestible (89.49% digestibility) in the initial intestinal phase. The entire digestion involved IgY aggregation, degradation, re-aggregation, and gradual decomposition into small pieces (by dynamic light scattering). These results indicated that IgY was impressionable, unstable and changeful in gastrointestinal environment, which might impair the bioactive function of IgY. Over 6 peptides (such as RGFK, TVPSGASTK, VPAATASPR) and 21 amino acids were detected, including 6 essential amino acids (methionine, isoleucine, leucine, histidine, tryptophan, and lysine), suggesting that IgY could be involved in human health regulation as active peptides or as rich sources of amino acids in addition to its own bioactive functions. The digestion kinetic curve confirmed that IgY did not reach its maximum digestion at the end of simulation of intestinal phase, implying the incomplete utilization of IgY. This study provides valuable details of oral IgY for development as active ingredients of a functional food, contributing to boosting the egg industry and improving human health.

Use of fungal and bacterial protease preparations to enhance extraction of lipid from fish roe: Effect on lipidomic profile of extracted oil.

The present study investigated the hydrolysis of protein in hoki roe homogenate using a HT (bacterial), a FP-II (fungal) protease preparations and Alcalase (bacterial) to enhance lipid yield extraction. The degree of hydrolysis was determined at various pH, temperature and time using casein and hoki roe. Total lipid extraction and lipidomic analysis was carried out following proteolysis of hoki roe homogenate. The degree of hydrolysis and SDS-PAGE revealed that the hydrolytic capability of Alcalase was better than HT and FPII. The total extracted lipid yield was better following hydrolysis with Alcalase (19.29 %), compared to HT (18.29 %) and FPII (18.33 %). However, the total phospholipid (PL) and n-3 fatty acid yields were better from HT hydrolysed hoki roe homogenate (PL = 30.7 µmol/g; n-3 = 10.5 %), compared to Alcalase (PL = 22 µmol/g; n-3 = 5.95 %). Overall, this study indicates that HT protease preparation hydrolysis of fish roe homogenate can both enhance lipid extraction and retain lipid quality.

Effect of Limited Enzymatic Hydrolysis on Structural and Functional Properties of Elaeagnus mollis Oil Meal Protein.

Elaeagnus mollis oil (EMO) meal, a by-product of oil production with plentiful protein, is considered a cheap and good quality source of plant protein for use in the food industry. In this study, the influence of limited enzymatic hydrolysis of EMO meal protein on the structure, solubility, foaming and emulsifying capacities was investigated in detail. The hydrolysates with different DH values (5, 10, 15, and 17) were obtained by controlling the time of enzymatic hydrolysis with alcalase. The results showed that enzymatic hydrolysis decreased molecular weight and increased flexibility and surface hydrophobicity. At the given range of pH and concentration of NaCl, the solubility, foaming and emulsifying capacities of hydrolysates were significantly improved, especially in the area of isoelectric point, and increased with the increase of DH. It was also found that the hydrolysate with DH10 had better foaming and emulsifying stability. In general, appropriate enzymatic hydrolysis could improve the functional properties in favor of their potential use as food ingredients.

Influence of the Degree of Hydrolysis on Functional Properties and Antioxidant Activity of Enzymatic Soybean Protein Hydrolysates.

Soybean protein hydrolysates were prepared using two proteolytic enzymes (Alcalase and Protamex) and the degree of hydrolysis (DH) and their functional and antioxidant properties were evaluated. The highest DH value was 20%, with a yield of 19.77% and protein content of 51.64%. The total amino acid content was more than 41% for all protein hydrolysates. The protein hydrolysates from Protamex at pH 2.0 had excellent solubility, emulsifying activity, and foaming capacity, at 83.83%, 95.03 m2/g, and 93.84%, respectively. The water-holding capacity was 4.52 g/g for Alcalase, and the oil-holding capacity was 4.91 g/g for Protamex. The antioxidant activity (62.07%), as measured by the samples' reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) and the reducing power (0.27) were the strongest for Protamex. An ABTS activity rate of 70.21% was recorded for Alcalase. These findings indicated a strong potential for the utilization of soybean protein hydrolysates to improve the functional properties and antioxidant activity of soybeans as well as their nutritional values.

Effects of ultrasound pretreatment at different powers on flavor characteristics of enzymatic hydrolysates of cod (Gadus macrocephalus) head.

To make better use of cod head (Gadus macrocephalus), a by-product of fish processing, the effects of ultrasound pretreatment on the enzymatic properties and volatile compounds of cod head enzymatic hydrolysates were investigated. The results demonstrated that ultrasound pretreatment at 150-600 W had a positive effect cod head enzymatic hydrolysates. The soluble peptides content of the enzymatic hydrolysates reached the highest value of 5.31 ± 0.16 mg/mL at the ultrasound power level of 450 W, and the content of peptide molecules < 3-kDa was up to 93.96%. The type and relative content of volatile compounds, especially aldehydes, alcohols, and ketones, also increased with the increase the ultrasound power. The electronic tongue results indicated that ultrasound pretreatment reduced bitterness and astringency. The electronic nose results indicated that the hydrogen- and alkane-containing odor components in the hydrolyzed liquid after ultrasound pretreatment differed significantly from conventional enzymatic hydrolysates. In conclusion, ultrasound pretreatment may be applicable as a suitable technology to assist enzymatic hydrolysis of the cod head, and as such, promote the utilization of fish by-products.

Whey Protein Hydrolysates of Sheep/Goat Origin Produced by the Action of Trypsin without pH Control: Degree of Hydrolysis, Antihypertensive Potential and Antioxidant Activities.

Tryptic WPHs with considerable residual whey protein content intact were developed from two sheep/goat WPCs (65% and 80% protein) without pH control. Pasteurization was used to avoid denaturation. Changes in non-protein nitrogen (DH_TCASN), free amino groups (DH_TNBS), and major whey proteins were used to investigate the degree and extent of hydrolysis. Antihypertensive potential (ACE-IA), radical scavenging (DPPH-RSA), and iron chelation (Fe-CA) were assessed. No statistically significant changes in pH (5.84−6.29) were observed during hydrolysis and storage. At the start of hydrolysis, DH_TCASN was ≅11% for both substrates whereas DH_TNBS was >10% and >5% for WP65 and WP80, respectively. After one-hour hydrolysis, DH_TCASN was ≅17% for both substrates and DH_TNBS was ≅15% and ≅11% for WP65 and WP80, respectively. The ß-lactoglobulin, α-lactalbumin, and caseinomacropeptide of WP65 were hydrolyzed by 14 ± 1.3%, 73.9 ± 2.6% and 37 ± 2.6%. The respective values for WP80 were 14.9 ± 1.7%, 79.9 ± 1%, and 32.7 ± 4.8%. ACE-IA of the hydrolysates of both substrates was much higher (>80%) than that of controls (<10%). Hydrolysis, substrate type, and storage did not affect the DPPH-RSA (45−54%). Fe-CA of the WP65 and WP80 hydrolysates were ≅40% and ≅20%, respectively; a similar outcome was found in the respective controls. Refrigerated storage for 17 h did not affect the degree of hydrolysis and biofunctional activities.