vol 21 no 2, p 1-9 antioxidative activity of camel milk ... activit… · 4 / december 2014 journal...

Journal of Camel Practice and Research December 2014 / 1

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Vol 21 No 2, p 1-9

ANTIOXIDATIVE ACTIVITY OF CAMEL MILK CASEIN HYDROLYSATES

Abdulrahman A. Al-Saleh1, Ali A.M. Metwalli1,2, Elsayed A. Ismail1,3 and Omar A. Alhaj1,4

1Department of Food Science and Nutrition, College of Food and Agricultural Sciences,King Saud University, Riyadh, P.O. Box 2460, Saudi Arabia

2Department of Dairy Science, College of Agriculture, Minia University, Minia 61519, Egypt3Department of Food Science, Faculty of Agriculture, Benha University, Benha 13518, Egypt

4Research and Development, Iraqi Dairy Products and Ice Cream Company, P.O. Box 3278, Baghdad, Republic of Iraq

ABSTRACTAntioxidant activity of camel and cow milk hydrolysed casein was studied. Camel and cow milk casein were

hydrolysed using trypsin enzyme for 0 to 180 min. Total phenol compounds, reducing power, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity and lipid hydroperoxide were determined as indicators for antioxidative potential of hydrolysed casein. Total phenol compounds; reducing power and DPPH scavenging activity of camel milk casein hydrolysate were significantly (P < 0.05) higher than those of cow milk casein hydrolysate. However, the lipid hydroperoxide formation was significantly (P < 0.05) lower in camel milk casein hydrolysate than in cow milk casein hydrolysate. The study indicated that the camel milk hydrolysate had higher antioxidative potential than cow milk casein hydrolysate. Moreover, the hydrolysis of caseins improved their antioxidant activity in both casein types.

Key words: 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), antioxidative potential, camel, casein, lipid hydroperoxide

Antioxidant compounds are considered to be essential in diets or medical therapies for delaying aging and biological tissue deterioration (Frankel, 1996). The search for natural antioxidants is of great interest both in industry as well as in scientific research (Lucia et al, 2002). Antioxidant peptides present in the food system play a major role in immuno-system by preventing the formation of free radicals or by scavenging free radicals and active oxygen species, which induce oxidative damage to biomolecules and cause aging, cancer, heart disease, stroke, and arteriosclerosis (Kadri et al, 2011; Gupta et al, 2009).

Antioxidants from natural sources are more superior to those produced chemically, because some synthetic antioxidants have been reported to be carcinogenic (Liu et al, 2005). A number of bioactive peptides have been identified in milk protein hydrolysates and fermented dairy products (Nagpal et al, 2011; Srinivas and Prakash, 2010; Kamau et al, 2010; Korhonen, 2009; Contreras et al, 2009). Cow casein consists of 4 casein fractions: αs1; αs2; β and κ- casein in ratio 4:1:4:1 (Fox and Mcsweeney, 1998) with phosphate content (mol/mol) 8-9, 10-13, 4-5 and 1-3, respectively.

Hattori et al (1998) reported that upon hydrolysis with certain enzymes or acids, some food

proteins can act as antioxidants in model systems. The ability of peptides to inhibit lipid oxidation appears to be related to certain amino acid residues in the peptides, such as tyrosine, methionine, histidine, lysine, and tryptophan, which are capable of chelating pro-oxidative metal ions (Diaz et al, 2003; Karel et al, 1966). Milk caseins may inhibit lipid peroxidation possibly through the mechanism of increasing iron autoxidation (Todoroki et al, 2005). Peptides from casein protein hydrolysate have been shown to possess superoxide anion scavenging activity (Cervato et al, 1999). The bioactive peptides derived from cow milk casein have been extensively studied (Ries, 2009; Korhonen, 2009). The aim of this study is to determine the antioxidative potential of camel milk casein hydrolysate in comparison with that of cow milk casein.

Materials and Methods

Enzymatic HydrolysisA 2.5% casein solution of camel or cow milk

casein was suspended in water at pH 7.0 using 1 mol NaOH. After complete casein solublisation, the temperature was adjusted at 37°C and the trypsin enzyme was added to casein (substrate) in ratio 1/100 and incubated for 0 to 180 min. The enzyme

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0.25 mL of the casein samples were reacted with 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent for 4 min, and then 2 ml saturated sodium carbonate solution (about 75 g/L) was added into the reaction mixture. The absorbance readings were taken at 760 nm after incubation at room temperature for 2 h. Gallic acid was used as a reference standard, and the results were expressed as milligram gallic acid equivalent (mg GAE)/100 ml casein solution sample.

Reducing powerThe Fe3+ -reducing power of the untreated

and treated camel and cow casein samples were determined by the method of Oyaizu (1986) with a slight modification. A 0.5 ml samples were mixed with 0.5 ml phosphate buffer (0.2 M, pH 6.6) and 0.5 ml potassium hexacyanoferrate (0.1%), followed by incubation at 50°C in a water bath for 20 min. After incubation, 0.5 ml of TCA (10%) was added to terminate the reaction. After centrifugation at 3000 g the upper portion of the solution (1 ml) was mixed with 1 ml distilled water, and 100 µl FeCl3 solution (0.01%) was added. The mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm. All experiments were performed 3 times. A greater reducing power was detected by a higher absorbance of the mixture. A positive control was butylated hydroxytoluene (BHT).

Determination of hydroperoxideOlive oil/gum arabic emulsion substrate was

prepared by combining 10 g each olive oil and gum arabic in a 400-ml beaker. Bring volume to 200 ml with 50 mM sodium phosphate buffer, pH 8.0, and homogenise 5 min using a domestic blender at a setting that does not cause excessive foaming. For the determination of lipid hydroperoxides, a method adapted from Shantha and Decker (1994) and Nuchi et al (2001) was applied. Casein hydrolysate was added to emulsion substrate in concentration 5% and incubated at 50°C for acceleration of lipid oxidation. One ml sample was withdrawn and kept in freezer until use. To extract fat from emulsion sample mixture, 5 ml of an extraction solvent (ethanol/ethyl acetate/n-hexane, 1:1:1 (V/V/V), was added to 1 ml of frozen emulsion aliquots, left thawing at room temperature for 20 min. and followed by vortexing and centrifugation. Supernatant (0.5 ml) was transferred to a reaction tube with 4 ml of methanol/butanol (2:1 (V/V). Thiocyanate (30 μL of 30%) and ferrous iron solution (30 μL) were added. After each addition, the reaction tubes were vortexed for a few seconds. Blank samples were prepared in

inactivation was achieved by heating the samples in boiling water bath for 5 min. Samples were cooled under running tap water and kept in the refrigerator until use.

Degree of hydrolysisDegree of casein hydrolysis was determined

using the ortho-phthaldialdehyde (OPA) according to Donkor et al (2005). The hydrolysis degree of samples was estimated using the equation:- (S-C)×100% degree of hydrolysis = D-C

Where S is the reading of casein hydrolysate samples by trypsin enzyme, D is the reading of hydrolysed samples for 24h incubation; C is the un-hydrolysed casein reading. Degree of casein hydrolysis was also determined by SDS-PAGE as described according to Laemmli (1970) using 12.5% acrylamide resolving gel and a 4.5% acrylamide stacking gel.

DPPH scavenging activity analysisThe antioxidant activities of camel and cow

milk casein hydrolysates were measured in terms of hydrogen donating or radical scavenging ability using the DPPH (Brand-Williams et al, 1995). The same method was adopted here to evaluate the antioxidant activities of camel and cow casein milk hydrolysate. DPPH solution was prepared as follow: 24 mg DPPH was dissolved in 100 mL methanol, this solution stored at -20°C as stock solution. Working solution was freshly prepared by mixing 10 ml stock solution with 90 ml methanol. 250 µl of sample was added to 4 ml DPPH solution. The mixture was shaken vigrously and then, absorbance measurements commenced at 517 nm after 30 min and 24h incubation. Methanol was used as blank and DPPH working solution was used as positive control. The per cent decrease in the absorbance was recorded for each sample, and per cent quenching of DPPH radical was calculated on the basis of the observed decrease in the absorbance of the samples. Antioxidant activity was calculated as follows:

% DPPH scavenging activity= (1 - [A sample ⁄ A control]) ×100

All DPPH tests were carried out in triplicates.

Determination of Total Phenolic ContentTotal phenolic content was determined with

the Folin-Ciocalteu reagent according to a procedure described by Singleton and Rossi (1965). Briefly,


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Where A is absorbance of sample; B is absorbance of blank and C is absorbance of oil emulsion without casein solution.

Statistical analysisData were analysed by the general linear

models procedure of SAS (1996) software. Analysis of variance (ANOVA) was done to determine the significance of main effects (type of casein and degree of hydrolysis). Significant (P < 0.05) differences between means were identified by Tukey mean test.

Results and discussion

Degree of Hydrolysis (DH)The DH of camel milk casein ranged from

53.36% at 5 min incubation to 79.24% at 180 min and for cow casein ranged from 52.53 to 82.52%, at the same incubation times. More than 50% of both caseins (camel and cow caseins) were hydrolysed after 5 min incubation with addition of trypsin enzyme. With extension of incubation time to 180 min, additionally, 30% of caseins were degraded (Fig 1). These results

were confirmed using SDS-PAGE, where no bands were observed after 5 and 30 min incubation (Fig 2). Salami et al (2008) found that a complete degradation of cow and camel milk αs- and β –caseins with addition of trypsin enzyme and incubation for 15 min.

Total phenolic content (TPCs)Indigenous phenolic compounds (PCs)

in milk are not thought to pose a health risk to humans and may in fact have some positive effects

Fig 1. Hydrolysis degree (%) of camel and cow milk caseins by trypsin enzyme for 180 min. at 37°C.

Fig 2. SDS polyacrylamide gel electrophoresis of camel and cow casein hydrolysed by trypsin enzyme at 37° C, Lane 1: native camel casein; Lane 2: camel casein hydrolysed for 5 min; Lane 3: camel casein hydrolysed for 30 min; Lane 4: native cow casein; Lane 5: cow casein hydrolysed for 5 min; Lane 6: cow casein hydrolysed for 30 min.


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between camel and cow milk casein hydrolysates at all incubation times (from 5-180 min). Martin et al, 2002 observed that the phenolic compounds greatly varied according to type of diet introduced to cow. The phenolic compounds ranged between 2 to 9 mg/L when the cow fed at dry and pasture diet respectively. Moreover, O’Connell and Fox (2001) reported that the phenolic profile of milk greatly change according to the type of feed. The concentration of ptaquiloside and norsesquiterpene reached up to 50 mg L while, genistein at up to ~30 μg L; equol at up to ~300 μg L and daidzein at up to ~5 μg L, all derived from clover. Moreover, Hassan et al (2013) reported that, the phenolic compounds such as chlorogenic acid, vanillin, rosemarinic acid, catechin, gallic acid, qurecetin and caffic acid interact with casein and whey proteins. Phenolic compounds appear to have positive correlation to aroma in milk and cheese, and the aroma strength of 4-methyl phenol > 4-ethyl phenol > phenol and they conjugate in milk as sulphates or phosphates and can be liberated by aryl salphatase or acid phosphates which they are present in milk (Kitchen, 1985).

DPPH radical scavenging activityDPPH is a stable free radical of 2, 2-diphenyl-1-

picrylhydrazyl with a deep purple colour whose reaction with other radicals, reducing agents, or compounds capable of hydrogen atom transfer

(O’Connell and Fox, 2001). Servili, et al (2011) added 100-200mg/L phenolic compounds to produce functional milk beverages. The occurrence of PCs in milk and dairy products may be a consequence of several factors, e.g., the consumption of fodder crops by cattle, the hydrolysis of proteins by bacteria, contamination of milk with sanitising agents, process-induced incorporation or their deliberate addition as specific flavouring or functional ingredients effects ( O’Connell and Fox, 2001). The total phenol compounds (TPCs) of camel and cow casein milk proteins are presented in Fig 3. The TPCs of unhydrolysed camel milk casein, were significantly (p < 0.05) higher than those of cow milk casein. The values were 4.30 and 4.99 mg phenol /100 g sample of cow and camel milk casein respectively. These results are in agreement with that obtained by Sreeramulu and Raghunath (2011) who reported that the concentrations of total phenolic compounds in toned milk (Indian fermented milk) and in buffalo milk was 3.4 and 3.0 mg/100g milk respectively. The concentration of TPCs significantly increased (p < 0.05) in both camel and cow milk casein hydrolysates after 5 min incubation ranged from 4.99 to 5.75 in camel casein hydrolysate to 4.30 to 4.97 mg phenolic compounds /100 g sample in cow milk casein hydrolysate. Slight increases in TPCs were found with increasing incubation times up to 180 min. Significant differences (p < 0.05) in TPC were found

Fig 3. Total phenolic content of camel and cow milk caseins hydrolysed by trypsin enzyme for 180 min. at 37°C.


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(HAT) leads to loss of colour at 515 nm The reaction continue more slowly (Apak et al, 2013). Though, the DPPH reactions were measured after 30 min and 24h incubation. Free radical scavenging activities of camel and cow milk casein hydrolysates are presented in (Fig 4). On degree of hydrolysis for 5 – 180 min

The DPPH scavenging activities of camel casein samples ranged from 27.05% to 36.82 % after 30 min incubation. The values for cow casein samples were ranged from 11.95% to 36.68% for 30 min of reaction. Significant differences (p < 0.05) in DPPH scavenging activity were found between unhydrolysed camel

Fig 4. DPPH radical scavenging of camel and cow milk caseins hydrolysates after 30 min of reaction.

Fig 5. DPPH radical scavenging of camel and cow milk caseins hydrolysates after 24h of reaction.


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and cow casein (27.05 and 11.95%, respectively). Salami et al (2011) reported that the hydrophobicity of β-casein is 0.339 to which the higher antioxidant activity. Therefore, the camel casein has considerable antioxidant activity even before it is hydrolysed. Taylor and Richardson (1980) found that the relative effectiveness of casein fractions as antioxidants were in the order β-casein (2.40) > κ-casein (2.20) > αs-casein (1.81). The percentage of β-casein in camel milk is about 65% (Elahj, 2010). DPPH scavenging activities increased from 27.05% to 34.36% for camel casein and from 11.95% to 33.73 % for cow casein hydrolysate (5 min incubation). No significant differences (p < 0.05) in DPPH scavenging activity were found between camel and cow casein samples with increasing incubation time from 5 min to 180 min. Moreover, the degree of hydrolysate had no significant effect on DPPH scavenging activity after 5 min incubation (Fig 4). DPPH scavenging activity for camel and cow casein hydrolysate significantly increased when the reaction mixture stored for 24 h. Most of DPPH scavenging activity occurred with 5 min hydrolysate in camel and cow casein hydrolysate. The values were increased from 28.23 to 71.51% in camel casein after 5 min incubation while it increased from 11.18 to 53.53% at the same incubation time for cow casein sample. With increasing the incubation time to 180 min, the DPPH scavenging activity increased to 81.68 and 63.70% for camel and cow casein hydrolysate, respectively (Fig

5). Cumby et al (2008) reported that DPPH scavenging activity of canola protein increased in protein hydrolysate in comparison with unhydrolysed protein. They attributed the increase of DPPH scavenging activity in protein hydrolysate to the appearance of functional groups of protein hydrolysate. The free radical scavenging ability of casein hydrolysates towards radicals from DPPH clearly significantly (p < 0.05) improved with increasing degree of casein hydrolysis. It was suggested that the increased oxidative stability of casein hydrolysates could have been due to a greater exposure of free radical scavenging amino acid residues (e.g. tryptophan, tyrosine, phenylalanine, cysteine or methionine) (Ries, 2009; Arcan and Yemenicioglu, 2007; Moure et al, 2006). Hoeller and Hassan (1965) reported that the amino acid contents of methionine, valine, phenylalanine, arginine and leucine are greater in Bactrian camel milk casein than in cow milk casein. However, the free radical scavenging activity of amino residues alone did not account for the whole free radical scavenging activity of peptides (Hernandez-Ledesma et al, 2005; Diaz and Decker, 2004). That means that the pool of amino acid-peptide mixture has higher free radical scavenging activity than either free amino acids or polypeptides. The same observation was confirmed by Salami et al (2011) who reported that the antioxidant activity of free amino acids were smaller than casein hydrolysates, which means that

Fig 6. Reducing power of camel and cow milk caseins hydrolysed by trypsin enzyme for 180 min. at 37°C.


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the primary structure of casein plays an important role. They also claimed that the most effective amino acids with antioxidant activity and the best free radical scavengers are Cys, Trp, Tyr, Met, Phe, His, Ile, Leu and Pro.

Reducing powerThe reducing power of a compound could

serve as an antioxidant potential activity indicator (Hazra, et al 2008). The higher absorbance of the reaction mixture indicated greater reducing power. As illustrated in fig 6 Fe+3 was reduced to Fe+2 in the presence of camel and cow casein hydrolysate and the reference compound butylated hydroxytoluene (BHT) to measure the reducing power. At 0° hydrolysate, (unhydrolysed samples) the absorbances of the camel, cow casein and BHT were 0.0.203; 0.038 and 0.02, respectively. These results indicated that camel and cow casein showed reducing power higher than those obtained by BHT. The results also revealed that the reducing power of camel casein at zero casein hydrolysate is significantly (p < 0.05) higher than that of cow casein. The reducing power of casein may be mainly attributed to presence of cysteine amino acid residues. Though, caseins have low levels of cysteine/ cystine and no free sulfhydryl groups. Only αS2 and κ- casein contain cystine (2 molecules/ casein molecule). Sulphur containing amino acids of bactrian camel milk is higher in particular methionine amino acids (Hoeller and Hassan, 1965). Moreover, the reducing power of casein hydrolysate significantly (p < 0.05)

increased with increasing the degree of hydrolysis in both types of caseins. The results from this parameter were in agreement with total phenolic contents (Fig 3) and DPPH scavenging activity (Figs. 4 and 5).

Percent decrease of hydroperoxide formationThe hydroperoxide concentration in bulk oils is

widely used to measure the deterioration of oil and fat. Presence of pro-oxidative metal ions such as iron; copper and cobalt in the emulsion mixture promotes lipid oxidation. However, the presence of effective chelators in the system makes metal ions unavailable for the lipid oxidation process. Milk proteins make as metal chelators (Ries, 2009). The effects of casein hydrolysates addition on decrease of hydroperoxide formation are shown in Fig 7. Results revealed that the formation of hydroperoxide decreased by about 6.47 and 4.97% with addition of unhydrolysed camel and cow casein, respectively. Ries (2009) found that the concentration of hydroperoxide decreased from 659 in control sample to 492 µmol/l with addition of sodium caseinate while it increased to 898 µmol/l with addition of water instead of sodium caseinate. The formation of hydroperoxide proportionally decreased with increasing sodium caseinate from 0.5 up to 10%. Addition of cow casein hydrolysate (incubation time for 5-45 min) had no significant (p < 0.05) effect on hydroperoxide formation and significantly decreased with increasing degree of hydrolysis (Fig 7). Camel casein hydrolysate significantly inhibited lipid hydroperoxide formation

Fig 7. Hydroperoxide decrease formation (%) of camel and cow milk caseins hydrolysed by trypsin enzyme at 37°C for 180 min.


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from 6.47 at 0° of hydrolysis to 11.61% at 5 min hydrolysis. Moreover, the formation of hydroproxide significantly decreased with increasing the degree of hydrolysis in both types of caseins. Addition of caseinophosphopeptides and casein hydrolysate inhibited lipid hydroperoxide and TBA reactive compounds production more than the unhydrolysed protein (Diaz and Decker, 2004). In a study of Diaz et al (2003), casein, tryptic casein hydrolysate and a low molecular weight fraction of tryptic casein hydrolysate (molecular weight ≤ 10 kd) were added to a corn O/W emulsion to test their influence on oxidative stability. They found that the stability to inhibit lipid hydroperoxide and hexanal generation generally decreased in the following order: low molecular weight casein hydrolysate > casein hydrolysate > casein. More aromatic amino acid residues (tyrosyl, phenylalanyl, and tryptophan) and sulphur containing amino acids became exposed with increasing degree of hydrolysis of the casein solutions. The increasing of antioxidative influence of the casein hydrolysates in the emulsion was attributed to the greater exposure of free-radical-scavenging amino acid residues (aromatic residues and free sulfhydryl groups), (Tong et al, 2000). Presence of non-oxidative cations such as sodium, calcium and magnesium may compete with iron ions for negatively charged binding sites on fat surface therefore, lower iron adsorption on the droplets decreasing lipid oxidation.

AcknowledgmentThe authors would like to extend their sincere

appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project no. RGP – VPP – 232.

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