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. 2021 Oct 9;59(7):2629–2642. doi: 10.1007/s13197-021-05282-3

Significance of whey protein hydrolysate on anti-oxidative, ACE-inhibitory and anti-inflammatory activities and release of peptides with biofunctionality: an in vitro and in silico approach

Chaudhari Hiralben Mansinhbhai 1, Amar Sakure 2, Ruchika Maurya 3,4, Mahendra Bishnoi 3, Kanthi Kiran Kondepudi 3, Sujit Das 5, Subrota Hati 1,✉
PMCID: PMC9207014  PMID: 35734133

Abstract

The study aimed to investigate potent antioxidant activities (ABTS assay, Hydroxyl free radical scavenging assay, and Superoxide free radical assay), ACE inhibitory activity, and anti-inflammatory activity in the WPC (whey protein concentrate) hydrolysate using Alcalase. The hydrolysis conditions (addition rate and incubation times) for peptide synthesis were also optimized using proteolytic activity. The generation of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages was reduced when the protein hydrolysate concentration was low. In comparison to unhydrolyzed WPC, SDS-PAGE examination revealed no protein bands in WPC hydrolysates. Two-Dimensional (2D) gel electrophoresis did not show any protein spots. Using the ‘In-solution trypsin digestion’ approach, the trypsin digested protein samples were put into RPLC/MS for amino acid sequencing. Peptides were also identified using RPLC/MS on fractions of 3 and 10 kDa permeates and retentates. The MASCOT database was used to look up the raw masses of LC/MS. By comparing hydrolyzed whey protein to the BLASTp (NCBI), PIR, BIOPEP, and AHTPDB databases, novel antioxidative and ACE inhibitory peptides were reported.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-021-05282-3.

Keywords: Whey protein concentrate (WPC), Hydrolysate, Antioxidant, ACE inhibitory activity, Alcalase, Anti-inflammatory, Bioactive peptides

Introduction

Whey proteins concentrate (WPC), being a natural valuable peptide source, provides considerable nutritional and health benefits for humans due to its high level of branched-chain amino acids, high protein content (30–80%) (Suthar et al. 2017; Whetstine et al. 2005) and presence of promising functional molecules including immunoglobulin, α-lactalbumin, lactoperoxidase, albumin, lactoferrin and caseinomacropeptide (del Mar Contreras et al. 2011). However, WPC is less studied as compared to whey protein (the crude, non-purified form) and whey protein isolate (higher protein content than WPC). Hydrolysis of these whey products would generate smaller peptide fragments with different bioactivities, such as antioxidative (Lin et al. 2012), antihypertensive (Morais et al. 2015), immunomodulatory (Wu et al. 2018), antithrombotic (Silveira et al. 2013), antimicrobial (Boyacı et al. 2016) and opiate properties (Madureira et al. 2010). When whey protein concentrates or isolates are treated with acids, enzymes, or heat, the intact form of protein breaks down into peptides and amino acids, leading to the formation of whey protein hydrolysates (WPH). These pre-digested forms of whey protein are effectively absorbed in the gut, and the hydrolysates that are produced through enzymatic hydrolysis using protease enzyme contains the identical amino acid composition to that of the concentrate and isolate; thus, on ingestion, they can rapidly increase the amino acid concentration in the plasma as compared to intact forms of protein (Morifuji et al. 2010). Utilization of whey protein has been associated with the beneficial impacts on human wellbeing, especially within the prevention and management of metabolic disorders conditions like, cardiovascular disease, type II diabetes, mellitus, obesity and hypertension (Hati et al. 2015). Peptides are produced by hydrolyzing intake proteins with highly proteolytic enzymes or by fermentation with microorganisms. Peptides are also produced from milk proteins using proteolytic enzymes or proteolytic lactic acid bacteria. Researchers have shown that limited enzymatic hydrolysis of whey proteins can result in improved functionality and biological activities (Pouliot et al. 2009; Spellman et al. 2009). Food industry uses several enzymes for food production. However, using particular enzymes for each food has a great influence for improving the food biofunctional qualities. Food manufacturers prefer enzymatic hydrolysis due to the easy supply of a large range of enzymes that are thought of safe and natural as well as high peptide yields. Several enzymes are currently used in the industry as food grade enzymes and are used with customised functionality and biological activity for the production of whey protein hydrolysates (WPHs).

To date, research in this field has primarily focused on peptides’ antioxidant and blood-pressure-lowering effects (Toldra et al. 2020). Peptides can act as antioxidants in many ways, including by inactivating reactive oxygen species, scavenging free radicals, chelating pro-oxidant transition metals, reducing hydroperoxides, and inhibiting linoleic acid oxidation (Nwachukwu and Aluko 2019). The number of food protein hydrolysates and antioxidant peptides have been found to exhibit antioxidant activity, especially in casein casein bovine milk (Li et al. 2013; Samaranayaka and Li-Chan 2011). Depending on the proteases used, WPHs have demonstrated a wide range of antioxidant activity in an iron-catalysed liposome oxidation system or a copper-catalysed liposome emulsion (Colbert and Decker 1991). Dryáková et al. (2010) recorded that the antioxidant activity of whey protein increased from 7.0–19.8 to 40.0–54.2% on hydrolysis with microbial proteases (alcalase, flavourzyme, protamex and neutrase) (Dryáková et al. 2010). β-lactoglobulin Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile peptides have higher radical scavenging activity than butylated hydroxy anisole (BHA) peptides (Ricci-Cabello et al. 2012). Peptides are effective in preventing or treating hypertension by inhibiting angiotensin-converting enzyme (ACE) activity; ACE is an integral part of blood pressure regulation and electrolyte homeostasis (Aluko 2015). From sour milk, lactotripeptides [(isoleucine-proline-proline (Ile-Pro-Pro)] and valine-proline-proline (Val-Pro-Pro) were reported (Nakamura et al. 1995). A dipeptide (Tyr-Pro) is found to contain the whey fraction of a yoghurt-like substance, which developed a major antihypertensive effect in spontaneously hypertensive rats (SHR) (Yamamoto et al. 1999). It was also reported that whey protein isolates and native hydrolysates with antioxidant and anti-inflammatory peptides, when added to human epithelial colorectal adenocarcinoma Caco-2 cells that was exposed to H2O2, both inhibited production of IL-8 and reactive oxygen species (ROS) (Piccolomini et al. 2012). This study serves to determine the optimized conditions to generate bioactive peptides from WPC hydrolysates using alcalase, that possess maximum proteolytic, ACE-inhibition and antioxidant activities followed by their ccharacterization through RPLC/MS. Furthermore, the anti-inflammatory effect of the whey protein hydrolysate on LPS induced inflammation in murine macrophage (RAW 264.7) cell line was also studied.

Materials and methods

Materials and enzyme

Whey protein concentrate (WPC) 70% was procured from Charotar Casein Company, Nadiad, Gujarat, India. Alcalase (EC No. 3.4.21.62) (endoproteases) was purchased from Sigma Aldrich and stored at 5 ± 2 °C.

Sample preparation

In distilled water, a 5.0% (w/v) WPC 70 solution was prepared and heated to 95 °C for 5 min to inactivate native proteases present in the solution and cooled to 60 °C. pH of WPC solution was adjusted to 8.5 with 1 [N] NaOH with slow stirring. The mixture was held at room temperature for 10 min. Hydrolysis was performed by adding 1.0, 1.5 and 2.0% alcalase for 6, 7 and 8 h (pH 8.5, 65 °C). After hydrolysis, the solution was heated at 90 °C for 5 min (for inactivation of the enzyme) and then cooled it. Afterwards, solutions were centrifuged at 14,000 rpm for 30 min at 4 °C (Eppendorf centrifuge, USA) (Peng et al. 2010). The supernatant was collected and filtered using a 0.22 μm syringe filter (Millex®-HV, MERK, Ireland) and the filtered supernatant was further tested for antioxidant, proteolytic and ACE inhibitory activities.

Assessment of proteolytic activity

The hydrolysis conditions (rate of addition and incubation periods) of the alcalase enzyme were optimized by the use of the OPA method (O-phthalaldyde) (Hati et al. 2015). The filtrate (200 μl) was added to a 3 ml OPA reagent and the Spectrophotometer (Systronics PC based double beam Spectrophotometer 2202, India) measured the absorbance of the solution at 340 nm after incubation at room temperature (20 °C) for 2 min. The degree of proteolysis during enzymatic hydrolysis was calculated using the O-phthaldehyde (OPA) method by measuring the release of free NH3 groups.

Production of peptides using Alcalase enzyme under optimized hydrolysis conditions

In accordance with the sample preparation as stated above, the hydrolysates were prepared by using optimized hydrolysis condition (2.0% rate of addition and 8 h of hydrolysis), the hydrolysed WPC samples were centrifuged at 14,000 rpm for 30 min at 4 °C (Eppendorf centrifuge, US). The supernatant (water soluble extract) was collected and filtered through a syringe filter of 0.45 μm (Millex®-HV, MERK, Ireland). Then supernatant was passed through 3 and 10 kDa ultrafilter membranes (Amicon®-ultra-15, MERK, Ireland) at 14,000 rpm for 30 min at 4 °C (Eppendorf centrifuge, US) and then permeates and retentates were then collected from the membranes. The supernatants (permeates and retentates) were again filtered through a 0.22 μm syringe filter (Millex®-HV, MERK, Ireland) and further tested for antioxidant, proteolytic, ACE inhibitory activities and RP-HPLC analysis.

Assessment of antioxidant activities

Supernatants were prepared as mentioned above. This different permeate and retentate samples were also analysed for Antioxidant activity using ABTS assay, Hydroxyl free radical scavenging assay and Superoxide free radical scavenging assay.

ABTS assay (2,2-Azino-bis (3-ethylbenzothaizoline 6-sulfonic acid) assay) of hydrolyzed whey protein solution

The ability of a compound to scavenge the stable ABTS radical was based on the radical scavenging capacity of whey protein hydrolysates with alcalase following the method adapted from Mann et al. (2015).

Hydroxyl free radical scavenging assay of hydrolysed whey protein solution

Antioxidant activity was conducted using the hydroxyl-free radical scavenging capability of peptides (Li et al. 2008).

Superoxide free radical scavenging assay of hydrolysed whey protein solution

This method focuses on the ability of peptides during the reaction process to scavenge O2-by producing a chromophoric compound (Liu et al. 2010).

Assessment of proteolytic activity

According to the sample preparation mentioned above, the proteolytic activity was carried out following the methods of Hati et al. (2015).

Assessment of ACE inhibition activity

The Angiotensin converting enzyme (ACE) Inhibition activity of the hydrolysates was calculated as a percentage by using Hippuryl-L-histidyl-L-leucine (HHL) as substrate (Pan et al. 2012).

Separation of peptides through RP-HPLC

For the improved resolution of peptides, WSE filtered was injected into the RP-HPLC (Shimadzu LC-20, Japan). The protocol was followed as per Ferreira et al. (2007) with some modification as the HPLC solvent composition of Eluent-A was 0.01% (v/v) of TFA in deionized water and Eluent-B was 0.01% (v/v) of TFA in a mixture of 80:20 acetonitrile and deionized water. Separation was performed at room temperature at a flow rate of 0.25 ml/min. Ultra-filtered 3 and 10 kDa membranes were used to obtain various permeate and retentate samples and injected them into the RP-HPLC to isolate peptides. The relative proteolytic activity (%rpa) was calculated according to the method of Vasiljevic and Jelen (2002).

SDS-PAGE and 2D gel electrophoresis analysis of hydrolysed WPC solution

SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) analysis was conducted by using 15.0% separating gel (Ghosh 2015). The water-soluble extracts of hydrolyzed WPC solutions were used as a sample. Unhydrolysed 5.0% WPC solution was used as control by dissolving in 2X loading buffer, followed by heating at denaturation temperature (90 °C) for 5 min in PCR vials. Hydrolysates were 2–3 times precipitated with acetone solution. Then, it was spinned for 3 min and supernatants were discarded. The Pellets were collected and dissolved by adding a 20 µl 2X loading sample buffer, followed by heating at denaturation temperature in PCR vials at 90 °C for 5 min. The samples were loaded in SDS-PAGE. For the purification of peptides from WSEs of hydrolysed WPC solution, two-dimensional gel electrophoresis was also performed as per the protocol stated by Yang et al. (2014)

Identification and characterization of purified peptides through RPLC/MS

Liquid chromatography

The column, ACQUITY UPLC-BEH-C-18 (2.1 × 50 mm, 1.7 µm, WATERS, UK) was used. At 65 and 4 °C the column and sample temperature have been preserved, respectively. Water was the mobile phase A and Acetonitrile (ACN) was B with 0.1% formic acid each. At a volume of 5 μl, the sample was injected at a flow rate of 0.5 ml/min. Hydrolysed WPC solution digested with trypsin (“In solution trypsin digestion”) procedure was adapted from Kinter and Sherman (2005) and samples of 3 and 10 kDa permeate were also analysed on LC/MS network.

Data processing and identification of peptides

LCMS data were searched in MASCOT database for the identification and characterization of peptides using the methods prescribed by Jakubczyk and Baraniak (2014); Tagliazucchi et al. 2015) with some modifications (i.e., peptide mass tolerances set: 1.8 and MS/MS tolerances: 0.8).

Assessment of the anti-inflammatory effect of protein hydrolysate on LPS induced inflammation in murine macrophage (RAW 264.7) cell line

Cell viability assay

The mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan was used to measure cell viability. RAW264.7 cells were seeded in Dulbecco’s Modified Eagles medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), and 1% Penicillin–streptomycin solution at a cell density of 1 × 105 cells/well in 96 well plate. Confluent cells were treated with 0.25, 0.5 and 1 mg/mL of the alcalase treated protein hydrolysate. After 24 h, supernatants were discarded and MTT (0.5 mg/mL) was added to each well and incubated for 4 h at 37 °C in dark followed by the dissolution of insoluble formazan crystals in 100 µL DMSO. The absorbance of the resultant solution was measured at 570 nm using a microplate reader (NANO Quant-infinite M200 PRO, Tecan life science).

Effect of protein hydrolysate on LPS induced inflammation in macrophages

RAW 264.7 cells were seeded in DMEM supplemented with 10% FBS, and 1% PS solution at a cell density 1 × 105 cells per well in 96-well plates followed by incubation for 24 h at 37 °C under 5% CO2. Post confluency, cells were treated with different doses of the alcalase treated protein hydrolysates (0.25, 0.5 and 1 mg/mL) alone or in combination with LPS (1 µg/mL) for 16 h. Nitric oxide production in the supernatants was determined using Griess reagent method. Furthermore, TNF-α, IL-6 and IL-1β levels in the supernatants was determined using ELISA kits as per the manufacturer’s instructions.

Statistical analysis

Each test was performed three times according to the results obtained as means (Average) ± standard deviations (SD). To analyse the findings and different data sets were used experimental designs and statistical instruments. However, Steel and Torrie measured the significant difference between the treatments at 5.0% level of significance and analysis of variance (ANOVA) (Steel and Torrie 1980). Data on the cell culture experiments were expressed as mean ± SEM; n = 5 in (A), n = 3 in (B), n = 3 in (C–E). One-way ANOVA followed by Tukey’s post hoc test was used for analyzing the statistical significance. * p < 0.0001 relative to the control, # p < 0.0001 relative to the LPS.

Results and discussion

Production of peptides under optimized hydrolysis conditions

The overall proteolytic activity of WPC hydrolysate with alcalase ranged from 16.56 (1.0% rate of addition at 6 h of incubation) to 22.25 mg/ml (2.0% rate of addition at 8 h of incubation) as shown in Table 1. The proteolytic activity of WPC hydrolysate with alcalase was observed to be significantly increased (p < 0.05) with the addition rates (1.0, 1.5 and 2.0%). At 2.0% (21.61 mg/ml) rate of addition, proteolytic activity was significantly higher than 1.5% (18.61 mg/ml) and 1.0 per cent (16.90 mg/ml) for all incubation periods (6, 7 and 8 h). Proteolytic activity was found to be highest at 8 h (22.25 mg/ml) of incubation and 2.0% rate of addition than 1.0 and 1.5% of rate of additions.

Table 1.

Effect of rate of addition and incubation period of WPC hydrolysate with alcalase on proteolytic activity (mg/ml) (OPA activity)

Rate of addition (%) 6 h 7 h 8 h
1.0 16.56 ± 0.01a 16.89 ± 0.03b 17.26 ± 0.04c
1.5 18.37 ± 0.05a 18.59 ± 0.04b 18.87 ± 0.01c
2.0 21.00 ± 0.04a 21.59 ± 0.08b 22.25 ± 0.07d
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*Values with different superscripts differ significantly (p < 0.05), Proteolytic activity (absorbance) Mean ± SD

WPC hydrolysate with alcalase has a different proteolytic activity, with various rates of additions and periods of incubation. The increase in proteolytic activity with different additive rates was directly linked to the amount of alcalase required amino acids for which the release of free NH3 groups varies with the time of incubation. Recently, few researchers measured the proteolytic activity of the hydrolysed WPC solution produced by alcalase and also noted that the highest proteolytic activity was 89.2% at temperature = 58.2 °C, E/S ratio = 2.5%, pH = 7.5 and hydrolysis time Min = 361.0 (Hussein et al. 2020). Farup et al. (2016) also evaluated the in vivo plasma amino acid appearance in humans with three different hydrolyzed whey proteins, there impact observed were: High degree of hydrolysis (DH = 48%), Medium DH (DH = 27%), and Low DH (DH = 23%) found by OPA method (Farup et al. 2016).

Different antioxidant activities of 3 kDa and 10 kDa permeates and retentates of WPC hydrolysed with alcalase

10 kDa retentate of the 5.0% WPC showed the highest antioxidant activity (87.88%), followed by 3 kDa retentate (86.77%), 10 kDa permeate (85.91%) and 3 kDa permeate (79.81%) after hydrolysis at 8 h at 65 °C. Table 2 shows that the antioxidant activity was at par with the various treatments for the different 3 kDa permeate and 3 and 10 kDa retentates. However, a significant difference was observed (p < 0.05) between 3 kDa permeate and other permeates and retentates. The hydroxyl free radical scavenging assay examines the relative ability of antioxidants to scavenge the aqueous phase of free radical produced (Li et al. 2008). Hydroxyl free radical inhibition was measured spectrophotometrically at 536 nm. Maximum hydroxyl free radical scavenging activity (84.31%) was shown in the 10 kDa retentate sample (84.31%), accompanied by a 10 kDa permeate (70.11%), 3 kDa retentate (69.91%) and 3 kDa permeate (58.18%) after hydrolysis with alcalase for 8 h at 65 °C in 5.0% WPC solution. It was observed from Table 2, that the hydroxyl free radical scavenging varied significantly (p < 0.05) with different membrane treatments (permeates and retentates). The superoxide free radical scavenging assay analyses the antioxidant’s relative ability to scavenge the aqueous phase of the free radical produced. The original pyrogallol (1,2,3-trihydroxybenzene) process, which was originally developed for superoxide dismutase, is now commonly used for spectrophotometrically calculate superoxide scavenging of other antioxidants at 320 nm (Panchal et al. 2020). Maximum superoxide free radical scavenging activity was demonstrated by 10 kDa retentate (54.04%), followed by 3 kDa retentate (53.67%), 3 kDa permeate (40.08%) and 10 kDa permeate (39.47%) after hydrolysed with alcalase for 8 h at 65 °C in 5.0% WPC solution. From the Table 2, it had been observed that, Superoxide free radical scavenging activity was differing significantly (p < 0.05) with different treatments (10 kDa retentate and 3 and 10 kDa permeates). 3 kDa retentate (53.67%) and 10 kDa retentate (54.04%) demonstrated superoxide free radical scavenging activity at par.

Table 2.

Antioxidant activities of WSE of whey protein hydrolysates

Fractions Antioxidant activity (ABTS assay) (%) Hydroxyl free radical scavenging activity (%) Superoxide free radical scavenging activity (%)
3 kDa P 79.81b ± 1.51 58.18d ± 2.69 40.08b ± 1.23
3 kDa R 86.77a ± 0.93 69.91c ± 1.64 53.67a ± 2.55
10 kDa P 85.91a ± 0.67 70.11b ± 2.43 39.47c ± 0.72
10 kDa R 87.88a ± 1.55 84.31a ± 0.99 54.04a ± 2.38
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Here, P = Permeate, R = Retentate, kDa = kilo Dalton, % Antioxidant activity ± SD. 3 kDa and 10 kDa stands for the fractionated samples derived after UF. Values with different superscripts differ significantly (p < 0.05), Proteolytic activity (absorbance) Mean ± SD

In previous studies, the antioxidant activity of whey protein hydrolysate from cheese whey was evaluated. An increase in antioxidant activity was observed in the WPH permeate (3 kDa) (1.96 ± 0.001 μmol Trolox mg-1 protein) (Athira et al. 2015). The hydrolysis of whey protein isolate (WPI) by alcalase improved the antioxidant activity in liposome model systems, (Peng et al. 2010). The peptides with the highest radical scavenging activity were between 0.1 and 2.8 kDa. Likewise, it was reported that hydrolysates were most effective in scavenging the ABTS++ were prepared by hydrolyzing whey protein concentrates using microbial proteases (alcalase) (Dryáková et al. 2010). In a study, antioxidant rich milk protein peptides were developed through microbial proteases viz. Aspergillus oryzae validase (Val), Bacillus licheniformis alkaline protease (AP), and Bacillus subtilis neutral protease (NP). Sequential ultrafiltration has fractionated the hydrolysates (3 kDa and 10 kDa ultra filtered membrane). The findings showed with the highest antioxidant activity in the UF fraction (< 3 kDa) of milk protein hydrolysates (Hogan et al. 2009). Previous studies have indicated that whey protein isolate (WPI) enzyme hydrolysates have potential to prevent oxidative deterioration in an iron-catalysed liposome oxidation system, and that the antioxidative potential of enzyme hydrolysates could be improved by heat treatment of the WPI prior to hydrolysis (Pena-Ramos and Xiong 2001). Our study showed greater radical-scavenging ability of 10 kDa retentate using different antioxidant assays (ABTS assay, hydroxyl free radical scavenging assay and superoxide free radical scavenging assay). The whey protein hydrolysate produced by alcalase could be employed in the food industry as an antioxidant to replace synthetic antioxidants. Most of the studies showed that maximum antioxidant activities were observed in UF fraction (< 3 kDa) of milk protein hydrolysates. But we observed maximum free radical scavenging activity in 10 kDa retentates due to use of different enzymes, times of incubations, optimum pH and temperatures.

Proteolytic Activity (mg/ml) of 3 kDa and 10 kDa permeate and retentate of WPC hydrolysed with alcalase (OPA activity)

10 kDa retentate showed the highest proteolytic activity (17.87 mg/ml), followed by 3 kDa retentate (13.12 mg/ml), 10 kDa permeate (10.97 mg/ml) and 3 kDa permeate (05.98 mg/ml) after 8 h alcalase hydrolysis at 65 °C in a 5.0% WPC solution. It was found from Table 3, that there was a significant difference in proteolytic activity (p < 0.05) with different permeate and retentate treatments. Further, there was a significant difference (p < 0.05) found with various permeate and retentate treatments. The proteolytic activity of 3 and 10 kDa retentate was higher than that of permeate.

Table 3.

ACE Inhibitory activities, Proteolytic Activity and relative proteolytic activity of WSE of whey protein hydrolysates

Fractions ACE inhibition (%) Proteolytic activity (mg/ml) Peptide production (% rpa)
3 kDa P 47.91d ± 5.40 05.98d ± 0.39 64.27c ± 1.60
3 kDa R 53.43c ± 5.50 13.12b ± 0.10 70.47b ± 1.30
10 kDa P 57.98b ± 6.90 10.97c ± 0.25 76.35a ± 2.13
10 kDa R 81.42a ± 2.13 17.87a ± 0.50 84.49a ± 3.98
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Here, P = Permeate, R = Retentate, kDa = kilo Dalton, % Antioxidant activity ± SD. 3 kDa and 10 kDa stands for the fractionated samples derived after UF. Values with different superscripts differ significantly (p < 0.05), Proteolytic activity (absorbance) Mean ± SD

Previously, the researchers evaluated modified spectrophotometric and pH–stat methods for determining the degree of hydrolysis (DH) of hydrolyzed whey proteins. For the second stage hydrolysis phase tangential-flow filter, the highest DH of retentate (29.74%) was recorded (with 3 kDa), while the lowest (12.81%) was recorded with the single-stage 10 kDa TFF membrane (Cheison et al. 2009). Ena et al. (1995) demonstrated that WPC hydrolysate obtained with Alcalase was characterized by a hydrolysis degree ranging from 14.5 to 18%. Spellman et al. (2003) also found a similar hydrolysis degree of WPC, i.e. 14% by using Alcalase 2.4 L. The percentage of peptide bonds cleaved is defined as DH, which is an essential feature for proteolysis. When whey proteins are hydrolyzed for clinical use, there should be no peptides left that are big enough to carry epitopes. This translates to a high DH. However, a high DH of the processing will result in a high osmotic load, which may generate bitterness. The functional characteristics of whey proteins may change depending on the DH when they are hydrolyzed (Yu et al. 2019). In a study by Yu et al. (2019), according to the hydrolysis curve, DH of the WPI hydrolysates was carefully monitored and the enzyme action was stopped when the desired DH reached. In the experimental protocols, all trials were performed with the aim at fixed DH, which enabled comparison between the performance of polymerized samples of low, medium and high DH. DH of the prepared six WPI hydrolysates were 2.5, 8.2, 14.1% for alcalase, and 2.3, 7.9, 14.6% for trypsin. However, in our study WPC hydrolyzed with alcalase produced maximum proteolytic activity (17.87 mg/ml) in the 10 kDa fraction.

ACE inhibitory activity of 3 and 10 kDa permeate and retentate of WPC hydrolysed with alcalase

10 kDa retentate showed maximal ACE inhibition activity (81.42%), followed by 10 kDa permeate (57.98%), 3 kDa retentate (53.43%) and 3 kDa permeate (47.91%) after alcalase hydrolysis in 5.0% WPC solution for 8 h at 65 °C. It was found from Table 3, that the inhibitory activity of ACE was significantly different (p < 0.05) with different permeate and retentate membrane treatments. In addition, there was a significant difference (p < 0.05) found in the treatment of different membranes.

Peptides and their bioactivity of whey protein hydrolysates from cheese whey with multiple enzymes were reported previously. As a result, protease S showed the highest proteolytic activity and the ACE inhibitory activity was IC50, 0.099 mg/mL, and second was alcalase (IC50, 0.121 mg/mL), while trypsin (IC50, 0.317 mg/mL) had the weakest effect (Jeewanthi et al. 2017). In a study, ACE inhibitory peptides of chia (Salvia hispanica) produced by enzymatic hydrolysis was detected. A protein-rich fraction of the chia (Salvia hispanica L.) seed was ultrafiltered through four molecular weight cut-off membranes (1 kDa, 3 kDa, 5 kDa, and 10 kDa) after hydrolysed with an alcalase-Flavourzyme sequential system. Inhibition in the five ultrafiltered fractions ranged from 53.84 to 69.31% and was highest in the < 1 kDa fraction (69.31%). However, in our study it was observed that in the 10 kDa fraction, WPC hydrolysed with alcalase produced maximum ACE inhibitory activity (81.42%). This result is in line with the work reported by Silvestre et al. (2012), in which the ACE-inhibitory activity of whey protein hydrolysates was affected by the type of enzymes as well as the degree of hydrolysis. Also, the increased level of E/S ratio and alkali pH resulted in high values of DH to indicate the release of potent peptides which further contributed towards the strong ACE-inhibitory activity. These findings are comparable to another work reported by van der Ven et al. (2002) which used the same sample of whey protein hydrolysate. In a similar study, the optimum conditions for Alcalase-hydrolysis of WPC to produce protein hydrolysates with dual biofunctionalities of angiotensin-I converting enzyme (ACE) inhibitory was reported (Hussein et al. 2020). These data suggest that both the hydrolysate and the single fractions can be considered as a valuable source of ACE-inhibitory peptides, with the promise of application in the formulation of functional foods or dietary supplements that could prevent or treat hypertension.

Relative proteolytic activity (%rpa) of 3 and 10 kDa permeate and retentate of WPC hydrolysed with alcalase through RP-HPLC

Under optimized hydrolysis conditions (2% rate of addition, 8 h of incubation and 65 °C) in 5.0% WPC-70 solution, peptide production of all permeates and retentates was determined by the RP-HPLC analysis. The production of peptides was measured with respect to the unhydrolysed WPC solution (control). The peptide production of 10 kDa retentate was also observed to be significantly higher than the production of 3 kDa permeate and retentate (Table 3). While, compared to other permeates and retentates, 3 kDa permeate exhibited the lowest peptide production (64.27% rpa). The production of peptides was at par with 10 kDa of permeate and retentate.

The relative proteolytic activity of WPC-70 and Calcium caseinate supplemented fermented milk was evaluated by Patel and Hati (2018). The production of peptides was optimized through three cultures of Lactobacillus: L. helveticus (V3), L. rhamnosus (NS4), and L. rhamnosus (NS6) supplementing with WPC70 and Ca-caseinate at the rate of 1.0, 1.5, and 2.0% in DTM (double toned milk). In comparison with V3 and NS4 cultures, the NS6 culture produced the highest peptides at 1.0 and 1.5% WPC70, i.e. 42.50 and 54.50% respectively. Figure 1 showed RP-HPLC chromatograms of unhydrolysed WPC solution (A) and WPC hydrolysed with alcalase (B). It was found that peptide production was maximum in WPC hydrolysed samples compared to unhydrolysed WPC solution from the figures of unhydrolysed WPC solution (control) and WPC hydrolysed with alcalase. The region under the curve of each peptide profile was evaluated as an indirect measure of the proteolytic activity and, as a result, significant differences were found in the production of peptides by alcalase under optimized hydrolysis conditions (2.0% rate of addition, 8 h of incubation and 65 °C).

Fig. 1.

Fig. 1

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RP-HPLC chromatogram of Unhydrolysed WPC solution (control) (a) and WPC hydrolysate with Alcalase (b)

SDS-PAGE analysis of water-soluble extract (WSE)

Hydrolysed sample as well as water soluble extracts (WSEs) obtained from WPC hydrolysed with alcalase were subjected to SDS-PAGE analysis (Fig. 2a) along with low range molecular protein ladder (M.W. 10–315 kDa) from Puregene. In contrast to the unhydrolysed WPC solution, no protein bands were observed in SDS-PAGE from WPC hydrolysed with alcalase. This reflects the hydrolysis impact of alcalase through proteolytic activity (Fig. 2a and b). Since the proteins were completely hydrolysed, 3 and 10 kDa permeates and retentate did not display any protein bands on SDS-PAGE.

Fig. 2.

Fig. 2

Fig. 2

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a Protein profile of permeate and retentate of WPC hydrolysed with alcalase as revealed by SDS-PAGE (1. Protein ladder; 2. Unhydrolysed WPC; 3. alcalase; 4. 3kDaP; 5. 3kDaR; 6. 10kDaP and 7. 10kDaR) whereas, P for permeate and R for retentate. b 2D Gel Electrophoresis pattern of Unhydrolysed WPC sample-A and Hydrolysed WPC with alcalase-B. c The Total ion chromatogram of WPC hydrolysed with alcalase from In-solution trypsin digestion (EMS to EPI scan in LC–MS). d LVLDTDYK identified from WPC hydrolysed with alcalase of its protein score on MASCOT software

In a study, the effect of hydrolysis of whey protein neutrase, alcalase, and papain on iron uptake by Caco-2 cells were stated. Two new molecular weight (MW) bands around and below 10 KDa occurred at tricine SDS-PAGE for neutrase hydrolysates, and one new molecular weight (MW) band below 10 KDa for papain hydrolysates. No bands have been detected by hydrolysates with alcalase (Ou et al. 2010). As the WPC hydrolysate with alcalase did not display any protein bands on SDS PAGE, this work confirmed our outcome. Immunoreactive properties after enzymatic hydrolysis of peptide fractions of cow’s whey milk proteins was reported earlier. Using SDS PAGE, hydrolysates were analysed. Slight reaction bands were observed in WPC hydrolysates produced with alcalase (Wróblewska et al. 2004). In our study, similar results were observed as WPC hydrolysed with alcalase showed extremely slight band as revealed by SDS-PAGE.

2D gel electrophoresis

To purify protein molecules based on isoelectric point and molecular weight, two-dimensional gel electrophoresis of the hydrolysed WPC solution was performed. The 2D gel electrophoresis was performed with a 7 cm (3–10 pH) IPG strip. 2D Gel electrophoresis of WPC hydrolysed with alcalase was analysed and no protein spots were detected compared to the unhydrolysed WPC solution (Fig. 2b). This shows the complete proteolytic hydrolysis of the WPC solution with alcalase. The native protein was fully hydrolysed by the alcalse enzyme and its maximal proteolytic activity was shown in the Fig. 2a–b.

Identification and characterization of purified peptides through RP-LC/MS

No protein spots were seen in the 2D SDS PAGE of WPC-70 hydrolysed with alcalase. In addition, the ‘in-solution trypsin digestion’ method was used. WPC hydrolysed with alcalase was subjected to RP-LC/MS analysis for peptide recognition after digesting with trypsin. Permeates and retentates of 3 and 10 kDa of hydrolysed WPC with alcalase were subjected to RP-HPLC for characterization of peptides. In this study, total 36 peptides sequences of WPC hydrolysed with alcalase (15 from ‘In solution trypsin digestion’ of WPHs and 7 from 3 kDa permeate, 5 from 3 kDa retentate, 5 from 10 kDa permeate and 4 from 10 kDa retentate) were obtained from LCMS (Table 4). Peptides sequences were matched with bovine whey protein (Bos taurus) database in Swissprot (BLAST tools) and PIR (Protein Information Resources). All the characterized peptide sequences from 3 and 10 kDa permeate and retentate as well as ‘In solution trypsin digested’ samples were matched on BIOPEP (Bioactive peptides) database to confirm the antioxidant activity and also matched with antihypertensive peptide database (AHTPDB) for confirming the ACE inhibitory activity. Figure 2c shows the total ion chromatogram of WPC hydrolysed with alcalase from ‘In-solution trypsin digestion’ (EMS to EPI scan in LC–MS).

Table 4.

Sequences obtained from ‘In solution trypsin digestion’ and HPLC samples [3 kDa (permeate and retentate) and 10 kDa (permeate and retentate)]

‘In solution trypsin digestion’ of WPC hydrolysates with alcalase WPC hydrolysates with alcalase
Enzyme Sequence RPHPLC Sample Sequence
Alcalase QLTK 3 kDa Permeate RRGVVRKSRE
FLSHK WVLEKKARRR
FWYGWCK RQGK
PEQSLACQCL KRKPRF
LSFNPTQLEEQC FGGR
SLLLVGIL
VGINYWLAHK PFRKP
LDQWLCEK
MHIRL GREK
IAEKTKITP 3 kDa Retentate MAFRGRRPEL
NAWTSSNYDK RGPPELYYDK
WYSLAMAAS NEARKYVRNS
VRTPEVDDE
GDVFIQYICK PMIDVQIKMT
LHQTGIV SDIPAKP
WPC hydrolysates with alcalase
RPHPLC Sample Sequence RPHPLC Sample Sequence
10 kDa Permeate MWVRTTL 10 kDa Retentates DFGHIQYVAAYR
GQLRFGG NGCTEPLGLK
LPRIE EERAVIK
LVLDTDYK DFGHIQYVAAYR
NPWIQVNLMR
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In the present study, sequences of FWYGWCK, LVLDTDYK, PEQSLACQCL, LSFNPTQLEEQC, MHIRL and WYSLAMAAS peptides were released by WPC hydrolysed with alcalase from ‘In solution trypsin digestion’ and were partly matched with antioxidant fraction of WYS (β-lg) (Hernández-Ledesma et al. 2007), DYK (β-lg) (Tian et al. 2015), WYSLAMAASDI, WYSLA, LAC, ACQ, QCL (β-lg) (Pihlanto 2006; Hernández-Ledesma et al. 2007; Tian et al. 2015), EQC, CQC (β-lg) (Tian et al. 2015), MHIRL, MHI (β-lg) (Pihlanto 2006; Tian et al. 2015) and WYSLA (β-lg) (Hernández-Ledesma et al. 2007), on BIOPEP databases, respectively and confirmed its antioxidant activity. LVLDTDYK identified from WPC hydrolysed with alcalase and its protein score on MASCOT software as depicted in Fig. 2d. Further, sequences of SDIPAKP (from 3kDa retentate), LVLDTDYK (10kDa permeate) and DFGHIQYVAAYR (from 10kDa retentate) were partly matched with WYSLAMAASDI, AASDISLLDAQSAPLR (β-lg) (Pellegrini et al. 2001) and DYK (β-lg) (Tian et al. 2015) and IPIQYVL, WYSLAMAASDI (β-lg) (Hernández-Ledesma et al. 2007; Pihlanto 2006) and confirmed its antioxidant activity.

Sequences of QLTK, FLSHK, PEQSLACQCL, VGINYWLAHK, WYSLAMAAS and VRTPEVDDE were produced by WPC hydrolysed with alcalase from ‘In solution trypsin digestion’ and partly matched with antihypertensive fraction of NIPPLTQTPV, TKIPA (β-lactoglobulin) (Van der Ven et al. 2002), TPVVVPPFLQP, LAHKAL (Whey protein) (Tavares and Malcata 2013), LKGYGGVSLPEW, GVSLPEW (β-lg) (Tavares and Malcata 2013), VGINYWLAHKHK (β-lg) (Tavares and Malcata 2013), LAMA (β-lg) (Tavares and Malcata, 2013) and VRTPE, TPEVDDEALE (β-lg) (Hernández-Ledesma et al. 2007; Kopf-Bolanz et al. 2014) on AHTPDB databases, respectively. Further, peptide sequences of MAFRGRRPEL (from 3 kDa retentate), LPRIE (from 10 kDa permeate) and DFGHIQYVAAYR (from 10 kDa retentate) were partly matched with LAMA (β-lg), ALPMHIR (β-lg) and ALPMHIR (β-lg) previously reported by Tavares and Malcata (2013) from whey protein and confirmed its antihypertensive activity. Using the BIOPEP and AHTPDB database, ‘In solution trypsin digestion’ of WPC hydrolysed with alcalase and HPLC samples were found to align with β-lactoglobulin protein fraction and confirmed their antioxidant and antihypertensive activity.

Alcalase treated protein hydrolysates showed anti-Inflammatory effect against lipopolysaccharide induced inflammation in murine macrophage cells (RAW 264.7)

MTT assay of alcalase have negligible effect on cell viability at 0.25 mg/mL dose (Fig. 3a). The viability of cells ranges from 80 to 96%. Alcalase treated protein hydrolysates at 0.25 mg/mL concentration showed a significant reduction in nitrite production without compromising cell viability (Fig. 3a–b). Hence, 0.25 mg/mL concentration were selected to perform the subsequent NO inhibition experiment and pro-inflammatory intermediates viz. TNF-α, IL-1β, and IL-6. LPS induced NO production was prevented by LPS treatment for 24 h on macrophages resulted in the production of higher levels of IL-1β, IL-6, and TNF-α. This was prevented by co-treatment with alcalase with LPS on the macrophages (Fig. 3c–e). Various anti-inflammatory peptides have been released by the proteolysis of food grade proteins. Anti-inflammatory activities of two tripeptides, VPP and IPP, exhibited therapeutic effects in an intestinal enterocolitis model (Jauhiainen and Korpela 2007). In a similar study, it was reported with eight anti-inflammatory peptides from alcalase treated whey protein hydrolysate. Out of eight peptides, DQWL exhibited inhibitory ability on the mRNA expression of IL-1β, COX-2, and TNF-α, and the release of IL-1β and TNF-α in LPS-induced RAW 264.7 mouse macrophages at concentrations of 10 and 100 μg/mL (Ma et al. 2016).

Fig. 3.

Fig. 3

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Effect of the alcalase on a Cell viability (MTT assay) of RAW 264.7 cells, b Dose dependency in NO production c TNF-α d IL-6 e IL-1β measured in supernatants of LPS-stimulated RAW 264.7 cells. Data are presented as mean ± SEM; n = 5 in (a), n = 3 in (b), n = 3 in (c–e) and evaluated by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.0001 relative to the control, # p < 0.0001 relative to the LPS, LPS- lipopolysaccharide

Conclusion

For the synthesis, purification, and characterization of antioxidant and antihypertensive peptides, WPC-70 was hydrolyzed with alcalase enzyme at the rate of 2.0% for 8 h at 65 °C. The bioactivities of distinct antioxidative and antihypertensive bioactive peptides with different whey protein peptides were validated using the BIOPEP and AHTPB databases. The study found that hydrolyzed whey protein may be the most potent source of antioxidant and antihypertensive peptides, and that it can be used as a functional ingredient in a variety of food compositions. Anti-inflammatory properties were also found in alcalse hydrolyzed whey protein hydrolysate. In addition, validation of antioxidative and antihypertensive peptides from hydrolysed whey protein using the selected alcalase concentration is needed to be studied on in vivo models using small animals and human subjects.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 132 kb) (132.4KB, docx)

Authors’ contributions

CHM: Methodology, Investigation, Formal analysis, Data curation, Writing- Original draft preparation. AS: Data curation; Formal analysis; Methodology; Project administration; Resources; Software; RM: Data curation; Formal analysis; Writing—review & editing; Methodology; MB: Data curation; Formal analysis; Writing—review & editing; Methodology; KKK: Data curation; Formal analysis; Writing—review & editing; Methodology; SD: Writing- Reviewing and Editing, Visualization. SH: Supervision, Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Validation.

Availability of data and material

All data generated or analysed during this study are included in this published article.

Declarations

Conflict of interest

All the authors declare that there is no conflict of interest.

Footnotes

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 132 kb) (132.4KB, docx)

Data Availability Statement

All data generated or analysed during this study are included in this published article.

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