Dromedary milk was obtained from a dromedary (Camelus dromedarius) herd belonging to the Livestock and Wildlife laboratory, Arid Lands Institute of Medenine, Tunisia. Fresh dromedary milk was defatted by centrifugation (5000×g, 30 min, 4 °C, centrifuge Sorvall Lynx 6000; Thermo Fisher Scientific, Waltham, MA, USA). Then, it was lyophilized in a freeze dryer (Christ Gamma 1–20; Martin Christ GmbH, Osterode am Harz, Germany) and kept at –20 °C.

Pepsin from porcine stomach mucosa and pancreatin from porcine pancreas were obtained from Bio Basic (Ontario, Canada). Trypsin from porcine pancreas, α-chymotrypsin from bovine pancreas, papain from Carica papaya and pronase from Streptomyces griseus were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.

Dromedary milk protein hydrolysis was performed as described by Oussaief et al. (16) with slight modifications. Dromedary skimmed milk was resuspended on protein basis at 2.5% (m/V) in ultrapure water and the pH of this solution was adjusted to the optimal pH value of enzymes: 2 for pepsin, 8 for trypsin, α-chymotrypsin, pancreatin and pronase and 6.5 for papain, as given by the manufacturer. The hydrolysis was started by adding the enzymes to proteins at a ratio of 1:100 (by mass). The temperature of the reactions was maintained at 37 °C using a shaking water bath (LSB-030S; Daihan Labtech Co., Namyangju-si, Republic of Korea) at 150 rpm. After hydrolysis for 6 h, the enzymes were inactivated by heating the samples for 20 min at 85 °C. Dromedary milk protein hydrolysates (DMPHs) were neutralized to pH=7 and centrifuged at 10 000×g for 15 min at 4 °C. Then, the supernatants were freeze-dried and kept at −20 °C for further use. Control samples containing undigested dromedary milk proteins (UDMP) undergo the same procedure as the hydrolysates but without the addition of enzymes.

Degree of hydrolysis (DH) was measured as described by Hoyle and Merritt (17). A volume of 1 mL of each dromedary milk protein hydrolysate was added to 1 mL of a solution of 20% (m/V) trichloroacetic acid (TCA) and the mixtures were incubated at 25 °C for 30 min. Then, the mixtures were centrifuged at 10 000×g for 10 min at 4 °C to obtain 10% (m/V) TCA-soluble fraction. The total protein content of both 10% (m/V) TCA-soluble fractions and samples of dromedary milk protein hydrolysates were determined by the method of Lowry et al. (18). The DH value was calculated as the ratio of the 10% (m/V) TCA-soluble protein to the total protein in the sample, expressed as a percentage.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli and Favre (19) using a 5% (m/V) stacking gel and a 15% (m/V) separating gel. Samples were dissolved in a sample buffer (62.5 mM Tris-HCl buffer (pH=6.8), 2% (m/V) SDS, 10% (V/V) glycerol, 5% (V/V) β-mercaptoethanol and 0.0025% (m/V) bromophenol blue) at 1:1 (V/V) ratio and boiled for 3 min at 100 °C. Volumes of 10 μL of samples at 2 mg/mL proteins were loaded in the gel. After electrophoresis, proteins were fixed with 12% (m/V) TCA for 20 min, stained with 0.1% (m/V) Coomassie Brilliant Blue R-250 solubilized in a mixture of 50% (V/V) ethanol and 2% (V/V) TCA for 1 h and destained by several washes in 30% (V/V) ethanol, 10% (V/V) acetic acid solution.

Molecular mass distribution was determined using gel filtration chromatography by a Nexera XR HPLC system (Shimadzu, Tokyo, Japan) on a Superdex^® Peptide PE 7.5/300 column (GE Healthcare, Uppsala, Sweden) as described by Dupas et al. (20). A volume of 50 µL from each sample, filtered through a 0.45-μm syringe filters, was injected into the column. Elution was achieved at a flow rate of 0.25 mL/min with 30% (V/V) of 0.1% (V/V) trifluoroacetic acid (TFA) in acetonitrile and 70% (V/V) of 0.1% (V/V) TFA in water during 120 min. The elution was controlled spectrophotometrically at 215 nm (Cecil CE 2041; Cecil Instruments Ltd, Cambridge, UK). The column was previously calibrated using standard proteins: cytochrome C (12 400 Da), aprotonin (6500 Da), substance P (1348 Da), glycine 6 (360 Da), glycine 3 (189 Da) and glycine (75 Da).

Reversed-phase high performance liquid chromatography (RP-HPLC) analysis was conducted using a Nexera XR HPLC system (Shimadzu) equipped with a C18 Omnispher column (250 mm×4.6 mm, 5 µm; GE Healthcare) as reported by Adt et al. (21). Samples were filtered through a 0.45-µm syringe filter (Millipore Corp., Billerica, MA, USA) and a volume of 50 µL of each filtered sample was loaded onto the column. Peptides were eluted with 0.1% (V/V) TFA in water for 10 min, followed by an 80-minute linear gradient from 0 to 50% (V/V) of acetonitrile in the presence of 0.1% TFA, then a linear gradient of 50 to 75% (V/V) acetonitrile in 0.1% (V/V) TFA during 10 min. The flow rate was 1 mL/min and the separation was monitored spectrophotometrically at 215 nm.

All experiments were conducted in triplicate. The results were statistically assessed using SPSS v. 22.0 (29). The one-way analysis of variance (ANOVA) was performed with Tukey’s test to determine the significance at 5% probability level.

In this study, various proteases were employed to hydrolyse dromedary milk proteins in order to assess the functionality of the generated protein hydrolysates. The extent of proteolysis in the hydrolysates was assessed by determining the degree of hydrolysis (DH). Fig. 1 shows the kinetic curves of dromedary milk proteins. During the first 2 h of hydrolysis, the DH increased rapidly, indicating that dromedary milk proteins contain many cleavage sites for the used enzymes. After that, the hydrolysis rate decreased, which might be a result of the reduction in available cleavage sites for the enzymes. The typical shape of hydrolysis curves was found previously for cow’s milk protein hydrolysates (30) and goat’s milk protein hydrolysates (31).

Pronase-treated hydrolysates showed the highest DH values, whereas pepsin-treated hydrolysates showed the lowest ones, at each time interval of hydrolysis. After 6 h of hydrolysis, DH values were 17.69, 25.41, 31.35, 39.48, 18.79 and 41.86% for DMPH treated with pepsin (Pep), trypsin (Try), α-chymotrypsin (Chy), pancreatin (Pan), papain (Pap) and pronase (Pro), respectively. Different DH values of milk proteins have been reported in the literature due to the variability of the specificity of the enzymes used and the amino acid sequence of proteins. Banach et al. (32) reported that the hydrolysis of cow’s milk proteins with pepsin for 12 h lead to a DH of 5.7%, whereas the hydrolysis of these proteins with papain for 3 h and trypsin for 1 h showed a DH values of 9.8 and 14.1%, respectively. The DH values of cow’s milk casein hydrolysates after 24 h of hydrolysis with trypsin, pancreatin and papain were 20.68, 20.91 and 22.06%, respectively (33). For dromedary casein hydrolysates, the DH was found to be 22 and 16% after 6 h of hydrolysis with α-chymotrypsin and papain, respectively (15).The DH values in the current study show that the used enzymes have a broad specificity on dromedary milk proteins compared with the above DH values.

The significant difference (p<0.05) in the DH among the hydrolysates of this study could be mainly due to the specificity of the used enzymes. Pronase has very broad specificity of action towards proteins since it contains several proteinases and peptidases from Streptomyces griseus (34). Pancreatin is a mixture of enzymes liberated by the pancreas and it also has a broad specificity, but it has a preference for Arg, Leu, Lys and Tyr (35). Papain cleaves the bonds of Lys, Arg and Phe. Chymotrypsin attacks at the carboxylic side of aromatic (Phe, Tyr and Trp) and long chain hydrophobic (Met and Leu) residues while trypsin splits peptide bonds in the carboxylic group of Arg and Lys residues. Besides, pepsin cleaves preferentially peptide bonds involving aromatic amino acids (36).

The SDS-PAGE profiles of UDMP and DMPH are shown in Fig. 2. In this study, dromedary milk proteins before hydrolysis were predominantly lactoferrin, camel serum albumin, caseins, camel whey basic protein and α-lactalbumin. The protein profile of the hydrolysates changed. Indeed, lactoferrin band was absent from all the hydrolysates except from those obtained with trypsin and α-chymotrypsin, in which some traces of this protein were still noticed. In addition, bands of camel serum albumin and caseins were totally hydrolysed by all the enzymes into fragments invisible in the gel. Kumar et al. (15) also reported that dromedary milk caseins were rapidly hydrolysed with α-chymotrypsin and papain owing to their open structure. Camel whey base protein band was observed only in pepsin-treated hydrolysates. Except pronase, all the used enzymes exhibited limited degradation ability of α-lactalbumin. Banach et al. (32) reported that the hydrolysis of cow’s milk proteins with pepsin, trypsin and α-chymotrypsin degraded partially α-lactalbumin and β-lactoglobulin, whereas the rest of the proteins were all degraded into fragments invisible in the gel. The resistance of α-lactalbumin to proteolysis was attributed to its compact globular structure which hides its cleavage sites (14). Overall, most of high-molecular-mass proteins were degraded after enzymatic hydrolysis. The differences in enzyme specificity towards proteins might cause variability in protein degradation. These results suggest that the highest proteolysis occurred with pronase and the lowest with pepsin, which is in agreement with the results obtained for DH.

Gel filtration chromatography was performed to determine the molecular mass distribution of peptides in each sample (Table 1). UDMP had the lowest amount of low molecular mass peptides below 1 kDa (23.15%). After hydrolysis, the level of peptides with molecular mass higher than 10 kDa decreased, while the level of low molecular mass peptides (<1 kDa) increased, which confirms the generation of shorter peptides during the enzymatic hydrolysis. The differences in molecular mass peptide distribution depended on the used enzyme. Besides, the content of low molecular mass peptides positively correlated with the DH of the hydrolysates. In fact, hydrolysate DMPH-Pro, with the highest DH, comprised the highest amount of peptides with molecular mass under 1 kDa (86.41%), followed by DMPH-Pan (82.02%). For the other hydrolysates, the amount of low molecular mass peptides below 1 kDa ranged from 46.22 to 68.38%. Amiot et al. (37) reported that the hydrolysis of cow’s milk protein using trypsin (DH=6%) resulted in hydrolysates containing 60% peptides with molecular mass under 3 kDa. In addition, these authors found that cow’s milk protein hydrolysates obtained with α-chymotrypsin (DH=6%) contained 80% peptides with molecular mass below 1.2 kDa.

RP-HPLC is a good method to investigate the hydrophobicity of proteins and peptides (21). The RP-HPLC elution profiles of UDMP and DMPH are visible in Fig. 3. A few peaks with low intensities were detected in the chromatogram of the UDMP, which indicated that they contained only a few peptides. After hydrolysis, numerous peaks were eluted between 20 and 80 min in the DMPHs, confirming the hydrolysis of dromedary milk proteins into several peptides. These results are in agreement with the DH measurements, SDS-PAGE patterns and molecular mass distribution. During proteolysis, the breakdown of every peptide bond releases two highly hydrophilic chemical groups. Subsequently, the hydrophobicity as well as the molecular mass distribution of the hydrolysates decreased compared to the native proteins (38). The hydrolysates comprised peptides with different hydrophobic and/or hydrophilic properties. In fact, both pepsin- and papain-treated hydrolysates showed the highest content of hydrophobic (high retention time) peptides. However, pronase-treated hydrolysates, with the highest DH, contained more hydrophilic (low retention time) peptides than the other hydrolysates.

Fig. 4 a reports the DPPH radical-scavenging capacity of UDMP and DMPHs, at various concentrations. Both UDMP and DMPH were able to scavenge DPPH radical in a concentration-dependent manner. DMPH possessed greater DPPH scavenging capacity than UDMP (p<0.05). These results demonstrate that enzymatic hydrolysis of dromedary milk proteins leads to the liberation of peptides able to scavenge the DPPH radical (39). Whatever the concentration tested, DMPH-Pap exhibited the greatest DPPH scavenging activity (p<0.05). IC₅₀, defined as the concentration of samples required to inhibit 50% of the initial DPPH radical activity, was the lowest in DMPH-Pap (3.47 mg/mL). IC₅₀ values were between 4.43 and 5.23 mg/mL for the rest of the hydrolysates. DMPH had higher DPPH scavenging activity than dromedary casein hydrolysates determined by Kumar et al. (15), who found that the DPPH scavenging activity did not exceed 40%. These findings confirm that DPPH scavenging activity of DMPH resulted not only from the proteolysis of casein but also from the proteolysis of whey proteins. Therefore, proteolysis of whole dromedary proteins provides greater DPPH scavenging activity than casein and whey hydrolysed independently.

Fig. 4 b shows the results of ABTS scavenging activity of UDMP and DMPH. All the samples had the ability to quench the ABTS also in a concentration-dependent manner. ABTS scavenging capacity was more pronounced in DMPH than in the UDMP (p<0.05). These findings are similar to those by Kumar et al. (15), indicating that dromedary casein hydrolysates had higher ABTS scavenging activity than intact dromedary caseins. DMPH-Pap had the lowest IC₅₀ value (0.417 mg/mL), while UDMP had the highest (0.967 mg/mL). The IC₅₀ values for the other hydrolysates were between 0.437 and 0.836 mg/mL. These results are in agreement with the hydrolysates from dromedary colostrum proteins (16). However, Oh et al. (40) reported higher IC₅₀ values for cow’s milk protein hydrolysates than those in the present study. These findings may be explained by the fact that dromedary milk possesses higher content of antioxidant proteins like α-lactalbumin and β-casein than cow’s milk (9, 14).

Both UDMP and DMPH scavenged more ABTS radicals than DPPH radicals. This difference could be due to the capacity of ABTS and DPPH radicals to diffuse in the reaction medium. ABTS is soluble in alcoholic and aqueous solutions and then it could easily react with peptides present in an aqueous medium. However, DPPH is soluble only in alcoholic solutions and may not reach readily the peptides in aqueous media. Subsequently, a high capacity to scavenge ABTS does not always implicate a high capacity to quench DPPH (41).

Fig. 4 c presents the ferric reducing antioxidant power (FRAP) of UDMP and DMPH at various concentrations. The FRAP of all the samples was concentration dependent; it increased when the concentration of the samples increased. At all the tested concentrations, UDMP exhibited lower reducing power than DMPH (p<0.05). The improvement of the FRAP of dromedary milk proteins after enzymatic hydrolysis might be due to the increased availability of peptides able to reduce iron(III) ions. At a concentration range from 5 to 20 mg/mL, DMPH-Pap, which exhibited the highest DPPH scavenging ability, had also the highest FRAP, while DMPH-Pep had the lowest FRAP. The difference in the FRAP among the hydrolysates might be assigned to the specificity of the used enzymes. Similar results were registered for dromedary and cow’s milk casein hydrolysates (15, 33).

Fig. 4 d illustrates the chelating activity of UDMP and DMPH. The chelating power of Fe²⁺ increased considerably after the enzymatic hydrolysis of dromedary milk proteins. This observation could be due to the formation of amino acids with high metal-binding capacity (42). The hydrolysates chelated iron(II) ions in a concentration-dependent manner. The iron(II) chelating activity of the hydrolysates obtained from cow’s milk proteins (1 mg/mL) prepared by Conway et al. (43) did not exceed 25.5%, while for DMPH (at the same mass concentration) it was in the range of 45.67-69.20%. Indeed, dromedary milk contains higher amounts of lactoferrin than cow’s milk, and this protein is known as a good iron chelator (11). Among the different hydrolysates, DMPH-Pan exhibited the lowest IC₅₀ value (0.54 mg/mL), followed by DMPH-Chy (0.61 mg/mL), while the highest one was obtained with DMPH-Pap (1.35 mg/mL) (p<0.05). The difference in the chelating capacity among hydrolysates could be due to the difference in their amino acid sequence and composition of peptides since they were generated using enzymes with different specificities.

Solubility of proteins is a significant attribute, which is required for use in many functional applications because it influences other properties like foaming and emulsifying capacities. Solubility of UDMP and DMPHs at different pH values is shown in Fig. 5 a. Interestingly, enzymatic hydrolysis enhanced the solubility of dromedary milk proteins. Solubility of UDMP was minimum at pH=4.0 (33.11%), which is near the isoelectric point of dromedary milk caseins, and increased below and above this pH value. However, solubility of DMPHs exceeded 89% over the entire measured pH range. DMPH-Pro, with the highest DH (41.86%) and lowest molecular mass peptides, exhibited a high solubility (>98%) at pH=4.0 to 8.0. The improvement of the solubility of protein hydrolysates could be explained by the reduction of the molecular mass of proteins and the increase of ionizable groups (amino and carboxyl groups), which could enable the protein to build hydrogen bonds with water (38).

The foaming properties of proteins depend on their capacity to migrate to the air-water interface in order to decrease the surface tension (44). Foaming capacity and foam stability of DMPH and UDMP are shown in Fig. 5 b. The hydrolysates had higher foaming capacity than UDMP (p<0.05). The highest foaming capacity was found in DMPH-Chy, DMPH-Pan and DMPH-Pap and it was 67.11, 68.33 and 69.44% respectively, whereas UDMP had the lowest foaming capacity (7.22%). Gani et al. (13) also indicated that enzymatic hydrolysis of cow’s milk proteins increased foaming capacity. Its enhancement after enzymatic proteolysis could be due to the generation of amphiphilic peptides which could migrate more rapidly to the air–water interface to encapsulate air particles. Foaming expansion after whipping was monitored for 30 min to study the foam stability. DMPH-Try showed the highest foam stability (23.06%). The foam stability decreased with time (p<0.05), which is explained by the fact that some peptides did not have the capacity to maintain stable foam (13).

Emulsifying properties of DMPH and UDMP reported in terms of EAI and ESI are observable in Fig. 5 c. The EAI indicates the capacity of a protein to form an emulsion, while ESI reflects the ability of an emulsion to maintain unvarying properties for a certain period (45). EAI were significantly higher (p<0.05) in the hydrolysates than in the UDMP. The highest EAI was obtained with DMPH-Try (28.18 m²/g). This could be due to the presence of high amounts of amphiphilic peptides in this fraction that possess a great flexibility at the oil/water interface, which results in a large surface area, and, enhance the formation of an emulsion. However, the lowest EAI value was found in DMPH-Pro, the hydrolysate with the highest DH (8.05 m²/g). This observation could be explained by the existence of small peptides, which are less efficient in emulsion stabilization (46). The ESI of the hydrolysates was in the range of 34.28–74.82 min and the highest value was found in DMPH-Pan (p<0.05). Enzymatic hydrolysis generates peptides, which are surface active owing to their hydrophobic and hydrophilic groups. Thus, the EAI and ESI of the hydrolysates were enhanced. Emulsifying properties of the hydrolysates were influenced by the properties of the generated peptides including size, amphiphilicity and flexibility (46). In the same context, improvement of the emulsifying properties of cow’s milk protein hydrolysates was found by Luo et al. (33).