Int J Pept Res Ther (2010) 16:715 DOI 10.

1007/s10989-009-9196-x

Bioactive Peptides from Bovine Milk a-Casein: Isolation, Characterization and Multifunctional properties
S. Srinivas V. Prakash

Accepted: 29 October 2009 / Published online: 1 December 2009 Springer Science+Business Media, LLC 2009

Abstract a-Casein group of proteins makes up to 65% of the total casein and consists of aS1- casein, aS2- casein and other related proteins. Among all the proteases employed, chymotryptic peptides showed maximum inhibition for angiotensin converting enzyme (ACE). The degree of hydrolysis and release kinetics of the peptides during chymotrypsin hydrolysis was compared with biological activity and the potent peptides fractions were identied. The crude fraction obtained after 110 min of hydrolysis shows multifunctional activities, like ACE inhibition, antioxidant activity, prolyl endopeptidase inhibitory activity and antimicrobial activities. This fraction was further puried by HPLC and sequenced by mass spectra. This fraction constituted peptides with molecular weights of 1,205, 1,718 Da respectively. The sequencing of peptides by MALDI-TOF MS/MS shows sequences QKALNEINQF and TKKTKLTEEEKNRL from a-S2 casein. Keywords Peptides Mass spectra Bioactive Enzymatic hydrolysis Antioxidant activity Bovine milk Casein Angiotensin converting enzyme inhibition Abbreviations ACE ACEI DH DPPH 1 Gen/h

IC50 MALDI TOF OPA PEP RP TFA

Concentration of inhibitor at 50% inhibition Matrix assisted laser desorption ionization time of ight O-phthaldialdehyde Prolyl endo peptidase Reverse phase Triuoroacetic acid

Introduction Bioactive peptides from food proteins represent a source of health enhancing components that may be incorporated into functional foods and/or used as nutraceuticals (Meisel and FitzGerald 2003). Although several animal and plant proteins contain potential bioactive peptides (or sequences in the protein) milk proteins are currently the main source of biologically active peptides (Dziuba et al. 1999). Many bioactivities of milk are latent and inactive within the protein sequence, requiring enzymatic proteolysis for release. Bioactive peptides can be produced in vivo following intake of milk proteins by gastrointestinal enzymes, or can be produced in vitro by the proteolytic enzymes (Adermann et al. 2004; Meisel 2004). These peptides are separated by HPLC methods and characterized by mass spectra. Peptides with different activities like ability to enhance calcium absorption (Reynolds 1992), opioid activity (Chiba and Yoshikawa 1989; Clare and Swaisgood 2000), hypertensive/ACE inhibitor activity (Ariyoshi 1993), immuno-modulating (Korhonen and Pihlanto 2003a, b), and anti-microbial activities have been isolated from milk proteins (Jost 1993).

Angiotensin converting enzyme Angiotensin converting enzyme inhibition Degree of hydrolysis 1-diphenyl-2-picrylhydrazyl Generations per hour (growth rate)

S. Srinivas V. Prakash (&) Department of Protein Chemistry and Technology, Central Food Technological Research Institute (A Constituent Laboratory of CSIR), Mysore 570020, India e-mail: prakash@cftri.com

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Many milk-derived peptides are multifunctional, i.e., specic peptide sequences have more than one biological activity (Meisel 2004). Some regions in the primary structure of caseins contain overlapping peptides sequences that exert different biological effects (Meisel 2004). These regions have been considered as strategic zones which are partially protected from proteolytic breakdown (Meisel and Bockelmann 1999; Migliore-Samour and Jolles 1988; Hata et al. 1998; Meisel 2004; Hou et al. 2003). Milk protein-derived bioactive peptides are health-enhancing components that can be used to reduce the risk of disease or to enhance certain physiological functions (Meisel 2004; Silva and Malcata 2005). The aim of the present work was to study the effect of specic proteolytic enzymes and its hydrolytic fractions for its releasing multifunctional peptides from a-casein. The peptides liberated from the digests were screened for various biological activities and the potential multifunctional peptides were isolated from bovine milk a-casein. The hydrolysates were evaluated for antibacterial activity on pathogenic bacteria, ACE inhibition, antioxidant activity, prolyl endopeptidase inhibitory activity.

Isolation of a-Casein The isolation of a-casein was done using urea differential precipitation method (Hipp et al. 1951). The procedure for isolation of casein involves the following steps. The fresh milk from cow was defatted and centrifuged at 8,000 rpm for 20 min at 12C. After centrifugation, the pH of supernatant was adjusted to 4.6 with 1 N HCl by slow stirring. The solution was centrifuged at 8,000 rpm for 30 min and acid casein was separated out as pellet. The acid casein was dissolved in 6.6 M urea. After dissolving, the casein was diluted to 4.6 M, and centrifuged at 3,500 rpm at 25C for 20 min and the pellet was separated out. The pellet was washed twice with distilled water. This pellet was redissolved in 6.6 M urea containing 0.15 M NaCl and diluted to 4.63 M urea. This diluted solution was centrifuged at 3,500 rpm at 25C for 20 min. The resultant pellet obtained was resuspended again in 4.7 M urea and the residue was separated out by centrifugation. The solution was then dialysed against distilled water to remove excess urea and freeze dried. The protein was analysed on SDS PAGE, aminoacid composition and SEC-HPLC to conrm its purity and found to be 98% pure. Hydrolysis of a-Casein a-Casein was hydrolyzed by aminopeptidase, carboxypeptidase, bacterial protease, fungal protease and chymotrypsin. For all enzyme reactions studied, the enzyme to substrate ratio was maintained at 1: 150, in 10 mM morpholine-TFA buffer, for 120 min at pH 7.0 and at temperature of 37C. The enzymatic hydrolysis was stopped by keeping at 95C, for 5 min followed by immediate freezing (Tauzin et al. 2003). The proteolytic activity of chymotrypsin and fungal protease was measured using denatured hemoglobin as substrate and found to be 5000-tyrosine U mg-1 protein and 75000-tyrosine U mg-1 protein respectively. Purication of Peptides The peptides were puried initially on Shodex Protein KW 803, (8 mm 9 300 mm), porous silica gel ltration column with 1,50,000 exclusion limit, (Showa Denko America INC, USA) mounted on a Waters HPLC system equipped with Millennium software (Waters, Milford, USA). The 10 ll of hydrolysates was injected and isocratic elution was performed at ow rate of 1.0 ml/min using 10 mM morpholine -TFA buffer, pH 7.8 (Tauzin et al. 2003). The peptides collected from gel ltration were again repuried on reverse phase C18 column (4.6 mm 9 150 mm column, pore size 5 l Waters Symmetry column) on Waters HPLC (Waters, Milford MA, USA) system equipped with

Materials and Methods The following chemicals, alpha casein (C6780), chymotrypsin (C4129), from bovine pancreas type II, peptidase (P7500) from porcine intestinal mucosa, triuoroacetic acid (T6508), pancreatin (P5575), from Hog pancreas, bacterial protease (P5147), from Streptomyces griseus, Type XIV, angiotensin converting enzyme, (A6778) from rabbit lung, angiotensin converting enzyme inhibitor (A0772), Hippuryl-Histidine-Leucine tetrahydrate (H1635), 2,2-diphenyl-1-picrylhydrazyl (43180), lactoferrin (L9507) from bovine milk, O-phthaldialdehyde (P0657), L-ascorbic acid (A92902),urea, sodium chloride, N-ethyl Morpholine, denatured haemoglobin, SDS, 2-mercaptoethanol, a-cyano-4-hydroxy cinnamic acid, glycerol, Hippuryl Histidine Lysine, were procured from Sigma Aldrich chemical company, St. Louis, MO, USA. Z-proprolinal was obtained from Bachem AG, Bubendorf, Switzerland. Fungal protease (Aspergillus oryzae) was obtained from Amano Pharmaceuticals, Japan. Carboxypeptidase, from bovine pancreas, was from Millipore Corporation, Freehold, NJ, USA. Acetonitrile, ethanol, methanol, perchloric acid, ethyl acetate were from Spectrochem Labs, Mumbai, India. Coomassie G250 dye was from Bio-rad laboratories, USA. Brain heart infusion broth and nutrient agar, were procured from Hi Media, Labs Pvt. Limited, Mumbai, India. Milk was obtained from a Jersey cow and repeatedly procured from the same cow from Mysore, India.

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Millennium version 32 software. The protein was detected at 215, 230 and 280 nm with a ow rate of 0.7 ml/min by Waters 2996 Photo diode array detector. A gradient system of A: 0.1% TFA/water and B: 0.01% TFA acetonitrile/ water (70/30 V/V) was used for elution. The column was initially washed with a gradient of 040% B for 8 min, and further the gradient was expanded from 0 to 100% in 18 min. Finally the elution was completed with 100% A up to 25 min. The peaks corresponding to peptides were collected and freeze-dried (Tauzin et al. 2003). Quantication of Peptides The degree of hydrolysis was estimated by O-phthaldialdehyde method (Church et al. 1983). The methodology involves preparing the reagent containing 25 ml of 100 mM sodium tetraborate, 2.5 ml of 20% SDS, 40 mg of OPA (dissolved in 1 ml methanol) and 100 ll of b-mercaptoethanol. The nal volume was made up to 50 ml with distilled water. The reagents were prepared before the experiment. To assay the proteolysis of milk proteins as substrates, a small aliquot containing 1050 ll containing 5100 lg protein was added directly to 1 ml of OPA reagent in a spectrophotometer cuvette, and the solution was mixed briey by inversion and incubated for 2 min at 25C and the absorbance was recorded at 340 nm. The values were then subtracted from the blank and were converted percent degree hydrolysis (Church et al. 2001). The peptides were quantied by rapid Bradford assay as described in Sedmak and Grossberg (1997). Identication of Peptides by Mass Spectroscopy The peptides were identied by MALDITOF (Bruker Daltonics, Bruker GMBH, Germany). The lyophilized peptides were dissolved in 10 ll acidied water (0.1% TFA in water). From this 2 ll was taken mixed with 2 ll of the matrix solution. The matrix solution used was 0.05 M a-cyano-4-hydroxy cinnamic acid in acetonitrile/ethanol (1:1). The 2 ll was transferred onto the plate and allowed to dry. The peptides were desorbed and ionized by nitrogen laser. The analysis of the peptides was done on ex Analysis software provided with the instrument. The instrument was internally calibrated with standard peptide mixture (RP pep mix, Bruker Daltonics, Bremen, Germany). The peptide sequences from the tandem mass spectra were generated by using MS-Seq program in protein prospector software package version 4.0.5 (http://prospector.ucsf.edu, University of California, San Franscisco, CA, USA). The parameters are given as follows: instrument for analysis MALDI-TOF, enzymechymotrypsin, missed cleavage is 1, and percentage error in molecular weight is 2.0 Da. The peptide is dened as hydrogen ending N-terminal and

free acid at the C-terminal. The database used was SwissProt database (Clauser et al. 1999; Miranda et al. 2004).

Bioassays Angiotensin Converting Enzyme Inhibition Assay ACE inhibition activity was determined in vitro as follows: The known concentration of the peptide in 15 (l was incubated with 25(l of 26 mU/ml ACE (dissolved in 50% glycerol) for 30 min. After incubation 110 (l of 10 mM Hippuryl Histidine Leucine dissolved in 0.2 M phosphate buffer containing 0.3 M NaCl, pH 8.3, was added into reaction medium. The total reaction volume was 150 (l. Incubation was done for 80 min at 37C. The enzyme was inactivated by addition of 110 (l of 1 N HCl. The reaction product was extracted with 1 ml ethyl acetate, taking 750 (l of the organic layer, which was evaporated to dryness. Hippuric acid was redissolved in 1 ml water and the absorbance was recorded at 228 nm. The angiotensin converting enzyme inhibitor from Sigma was assayed in a similar way for comparison (Srinivas and Prakash 2008). One unit of ACE activity indicate 1 lmole of hippuric acid from Hippuryl-His-Leu per min in 0.2 M phosphate buffer, 300 mM NaCl, pH 8.3 at 37C. Antioxidant Activity Assay The antioxidant activity of the peptides was checked by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. 0.3 ml of the peptide was taken (660 (M) and made up to 0.4 ml with 0.1 ml of distilled water. To this added 0.6 ml of 100 (M DPPH reagent in methanol. The reaction mixture was incubated for 20 min under dark and the reading was taken at 517 nm. The decrease in absorbance at 517 nm was taken as the antioxidant capacity of the peptide. L - ascorbic acid was taken as standard (Srinivas and Prakash 2008). Prolyl Endopeptidase Inhibition Assay Prolyl endopeptidase activity from bovine blood serum was measured using Z-pro-prolinal as the substrate. Assay samples were prepared by mixing 20 ll of bovine blood serum with 1,960 ll of 020 lM inhibitor in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4. After 30 min at 37C, the reaction was started by the addition of 20 ll of 5 mM Z-pro-prolinal in methanol. The formation of naphtylamine was continuously monitored by uorescence (46 min at 37C; excitation and emission were at 340 and 410 nm, respectively). Enzyme reaction controls were included in the assay. Three independent

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10

Int J Pept Res Ther (2010) 16:715

experiments were performed, and the average was calculated (Sorensen et al. 2004). Mineral Binding Assay Enzymatic hydrolysates and synthetic peptides were incubated in zinc concentrations between 1 and 10 mg/ml in buffer, for 1 h at 37C with gentle stirring. Samples were determined for bound zinc by atomic absorption spectroscopy (model AA-6701F, Shimadzu) in absorption mode at 213.9 nm, (Dashper et al. 2005). Antimicrobial Activity Studies For assay of antibacterial activity, enzymatic hydrolysates were dissolved in water and sterile ltered (Millipore, 0.22 l) before use. The assay was carried out in sterile honeycomb micro plates (Honeycomb 2, Thermo labsystems, USA), and each well contained a total volume of 375 ll. 300 ll of freshly inoculated bacteria was added to each well, together with 75 ll of hydrolysates. The nal concentration of the hydrolysates in the wells varied between 0.75 and 3.0 mg/ml. Growth controls contained 375 ll of sterile growth media. The samples were incubated at 37C for 24 h. The absorbance (600 nm) was measured every 15 min by a plate reader (Bioscreen C, Growth Curves USA., New Jersey, USA). The experiments were repeated three times for each bacterial strain, with three parallels of each sample. The growth rate was calculated based on the Almaas et al. 2006. Statistical Analysis Standard deviation was estimated for all the spectrophotometric absorbance measurements on the various bacteria, based on parallels of each sample. Data were presented as mean values with calculated standard deviation. The results obtained from the different concentrations of hydrolysates were also analysed in order to investigate the signicance of difference. Students t-test (two-sample, assuming unequal variances) was run to compare the different growth curves and the differences were found to be (P \ 0.05) signicant.

hydrolysates of a-casein. It shows, the DH of 0.7% with aminopeptidase, 0.4% with carboxypeptidase, 4.6% with chymotrypsin, 4.2% with bacterial protease and 70% with fungal protease respectively. The DH with aminopeptidase and carboxypeptidase is less as they hydrolyze proteins from the N and C-terminal ends. Due to their narrow specicity, they are less productive in liberating potential bioactive peptides. The DH was more with bacterial and fungal protease due to their broad specicity of action. Fungal protease is having a mixture of endo and exopeptidase activity, which could be the reason for the increase in DH (Adler-Nissen 1986). Chymotrypsin is selected for its specicity to hydrolyze peptide bonds containing aromatic and hydrophobic amino acids (Adler-Nissen 1986). Chymotrypsin hydrolysates show 4.6% DH. Amiot et al. (2004) has shown that hydrolysis of whole milk with chymotrypsin shows DH of 4.8%. The DH with chymotrypsin is less and is perhaps due to the inhibition of enzyme by peptides or peptidepeptide interactions (Adler-Nissen 1986; Pouliot et al. 2000). Most bioactive peptides so far identied from milk hydrolysates contain four to ten amino acids (Xu 1998; Miller et al. 2000). Amiot et al. (2004) has shown that 80% of the peptides released by chymotrypsin appear to be in the range of 300 1,200 Da. Thus the chymotrypsin hydrolysates were selected for further study as they can liberate smaller peptides. Dunn and Lewis (1921) reported a comparative study on caseins with various proteolytic enzymes. They used pepsin, trypsin and erepsin enzymes and compared the hydrolysis pattern of the casein. But characterization of the liberated peptides by these enzymes are not investigated in detail for their multifunctionality.

75

a
Degree of hydrolysis (%)
60

45

b c

Results and Discussion Proteolytic enzymes are employed to generate peptides from a-casein. They are aminopeptidase, carboxypeptidase, fungal protease, bacterial protease and chymotrypsin. Peptides, liberated were collected and characterized. Estimation of DH was performed for each enzyme hydrolysates. Figure 1 compares DH proles of enzymatic

d e
0 40 80 120

Hydrolysis time (min)

Fig. 1 Comparison of degree of hydrolysis of enzymatic hydrolysates of a-casein. a Fungal protease b bacterial protease c chymotrypsin d carboxypeptidase and e aminopeptidase

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11

ACE inhibiting activity

80

ACE Inhibiting activity (%)

60

Percentage change

60

40

40

6
Degree of hydrolysis

20

0 0 a b c d e 0 20 40 60 80 100 120 140

Proteases
Fig. 2 ACE Inhibitory activity of the hydrolysates obtained from a-casein by using various enzymes a aminopeptidase b carboxypeptidase c fungal protease d bacterial protease and e chymotrypsin

Time of hydrolysis (min)

Fig. 3 Comparison of ACE inhibiting activity and the degree of hydrolysis of chymotrypsin hydrolysates. a Release kinetics of ACEinhibiting peptides from a- casein at different time intervals. b The degree of hydrolysis of a -casein

In order to isolate potential bioactive peptides from the enzymatic hydrolysates, they were initially screened against ACE inhibitory activity. ACE inhibitory activities of different enzymatic hydrolysates are shown in the Fig. 2. From the Figure, the chymotryptic peptides were having 64% 3 inhibition, whereas the enzymatic hydrolysates from aminopeptidase, carboxypeptidase, bacterial and fungal protease were found to be inhibiting ACE at 0, 4, 52 and 61% respectively. The ACE inhibition was found to be maximum for chymotryptic hydrolysates. The chymotrypsin hydrolysates were further studied for other biological activities like antioxidant activity, prolyl endopeptidase inhibitory activity and antibacterial activity. Peptide fractions after hydrolysis with chymotrypsin were collected at different time intervals and they were puried by HPLC. The hydrolysates were separated from unhydrolysed protein and the mixtures of several peptides were tested for the ACE inhibitory activity. Figure 3 shows the comparison of ACE inhibition and degree of hydrolysis of the liberated peptides, from a-casein at different time intervals. From the gure, it is evident that during the rst 10 min of hydrolysis, the ACE inhibition of the liberated peptides was found to be high. The ACE inhibition was 85% within the rst 10 min of hydrolysis, which reduced, to 30% after 25 min of hydrolysis time. From 25 to 150 min the ACE inhibition activity of the hydrolysates increases as a function of hydrolysis time from 35 to 65%. The fractions at the 105 min were taken for further studies, as their ACE inhibiting activity had reached plateau (65%). These fractions were considered for isolation of peptides, as there would not be any further hydrolysis and no subsequent change in DH%. The peptides released during the

rst 10 min of hydrolysis were not considered as further hydrolysis into smaller peptides were in the process. The study on release kinetics of peptides will help us to understand the optimum time required for hydrolysis to get potential bioactive peptides. Release kinetics of peptides was studied for the structural elucidation of (S2 casein (Tauzin et al. 2003). The 105 min fraction was analyzed for the multifunctional biological activities. Activities like ACE, prolyl endopeptidase inhibitory, antioxidant and antimicrobial activities against bacteria like Bacillus cereus (gram positive), Escherichia coli (gram negative) and Lactobacillus acidophilus (probiotic bacteria) were measured. The Table 1 shows multifunctional activity of the crude peptides. The crude peptides show more than one activity along with ACE inhibition. The IC50 values for ACE inhibition was found to be 0.1 mg/ml as compared to standard ramipril with IC50 values of 0.025 mg/ml. Ramipril is a well known orally administered hypotensive drug that acts on the ACE enzyme. IC50 values for PEP inhibition was 1.3 mg/ml as compared with the standard, Z-prolyl prolinal with an IC50 of 0.165 mg/ml. The peptides were found to be scavenging the oxygen free radicals with IC50 values of 1.25 mg/ml as compared with the standard, L-ascorbic acid of 0.175 mg/ml. These peptides did not show cytotoxic activity, as studied by the agglutination of red blood cells. The presence of antibacterial activity in contrast to cytotoxic activity could be due to the penetration of peptides into negatively charged bacterial membranes compared to neutral nature of eukaryotic membranes of red blood cells. The peptides show zinc binding activity. These peptides have zinc-binding capacity

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12 Table 1 The biological activity of chymotryptic hydrolysates and IC Sl. 1. 2. 3. 4 5

a b c

Int J Pept Res Ther (2010) 16:715 values Standard Ramipril L-Ascorbic acid Z-prolyl prolinal Bacitracin NAD IC50 0.025 0.001 mg ml-1 0.175 0.01 mg ml-1 0.165 0.02 mg ml-1
b

50

Biological activity ACE inhibitory activity Antioxidant activity (DPPH) PEP activity Zn2? binding activity Agglutination of RBC

IC50 0.1 0.02 mg ml-1 1.25 0.2 mg ml 1.3 0.5 mg ml NAD

c -1 -1

2.3 1.0 mg g-1a

45.0 2 mg g-1

NAD

Per gram of hydrolysates Values taken from Ming and Epperson (2002) No activity detected

of 2.3 mg/g of hydrolysate compared with the bacitracin standard of 45 mg/g as reported by Ming and Epperson (2002). The bioactivities of peptide are compared with the respective standards. The IC50 values of peptides are much higher as compared to the standards. This could be due to the presence of different nature of peptides in the hydrolysate. The higher IC50 values compared to standard IC50 values could be due to the peptide - peptide interactions. The reasons for the decrease in activity could also be due to the dilution of peptides in the mixtures (Meisel 1993; Meisel and Bockelmann 1999). This study is an indication of the presence of multifunctional activities in hydrolysates. The antimicrobial activity of the hydrolysates was studied using the bacterial growth curve method. Growth of Escherichia coli, Bacillus cereus and Lactobacillus acidophilus were followed in presence of hydrolysates and were compared with lactoferrin as standard. The growth rate of bacteria in presence and absence of hydrolysates and lactoferrin were calculated and are shown in the Table 2 for comparision. The growth rate of Bacillus cereus as control shows 2.4 gen/h as compared to presence of hydrolysates (0.75 mg ml-1) of 1.2 gen/h. In the presence of lactoferrin (0.75 mg/ml) the generation time of Bacillus cereus changed from 2.4 gen/h to 1.4 gen/h. The results showed that the hydrolysates and the lactoferrin have similar effect on the Bacillus cereus. Similarly the growth rate of Escherichia coli was 0.96 gen/h and in the presence of hydrolysates (0.75 mg/ml) the growth rate has reduced to 0.45 gen/h. The presence of lactoferrin growth rate of Escherichia coli has changed from 1.2 gen/h to 0.72 gen/h. The lag in the growth rate shows hydrolysates are more potent than the lactoferrin. The above study shows that the hydrolysates are effective on both Gram positive and Gram-negative bacteria. The growth rate for the L. acidophilus is 0.04 gen/h and in the presence of hydrolysates increases from 2.7 gen/h.

Table 2 The antibacterial activities of hydrolysates Concentration (mg ml-1) Growth rate (DOD600 Hydrolysates Bacillus cereus Control 0.75 1.5 3 .0 Escherichia coli Control 0.75 1.5 3 .0 Lactobacillus acidophilus Control 0.75 1.5 3 .0 0.04 (0.02) 2.7 (0.2) ND ND 0.04 (0.01) 0.04 (0.01) 0.04 (0.01) 0.04 (0.02) 0.96 (0.03) 0.45 (0.02) 1.14 (0.1) 1.14 (0.1) 1.2 (0.1) 0.72 (0.02) 0.9 (0.01) 1.02 (0.1) 2.4 (0.2) 1.2 (0.1) 2.4 (0.2) 1.8 (0.1) 2.4 (0.1) 1.2 (0.1) 1.8 (0.2) 1.8 (0.1)
nm)

gen/h (SD)

Lactoferrin

Students t-test: (P \ 0.05) signicant Lactoferrin is a standard antimicrobial protein from bovine milk

This indicates that the peptide promotes the growth of probiotic L. acidophilus while inhibiting the pathogenic bacteria (Table 2). The lactoferrin does not have any effect on the growth of probiotic bacteria. Students t-test (twosample, assuming unequal variances) were run to compare the different growth curves and the differences were found to be (P \ 0.05) signicant. In order to identify and characterize peptides, from hydrolysates, isolation and purication was undertaken. The peptides were puried by size exclusion chromatography using analytical HPLC system. The size exclusion chromatography prole of the 105 min fraction is shown in Fig. 4a. Different fractions were collected at different retention time intervals in the order of elution and each of the fractions were again checked for their ACE inhibitory

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Int J Pept Res Ther (2010) 16:715 Fig. 4 a Size exclusion chromatography prole of chymotrypsin hydrolysates. The peptides were separated in 0.01 M morpholine TFA buffer pH 7.8. The ow rate was maintained at 1.0 ml min-1. b Reverse phase HPLC pattern of chymotrypsin hydrolysates. The peptides were separated by C18 reverse phase column using binary gradient with a ow rate of 0.7 ml min-1. The arrow indicates the peak used for further separation

13

pattern. The peak collected at the retention time of 21.2 min shows ACEI of 98%. This fraction was collected and repuried on a reverse phase chromatography. Reverse phase column chromatography prole of this group of peptides eluted from 21.2 min peak is shown in Fig. 4b. Fractions were collected at different retention time intervals and checked for their ACE-inhibitory activity. The peak collected at the retention time of 15 min shows ACEI activity of 99.6%. The peptide fraction obtained at 15 min was characterized using mass spectra and shown in the Fig. 5a. The ngerprinting of the 15 min fraction shows 9 peptides with different molecular masses. The peptides that match with the alpha casein were selected for further the sequencing and characterization. The peptides with molecular masses of 1,204 and 1,718 Da were used for the MS/MS analysis as shown in the Figs. 5b and c. The amino acid sequences of the peptides from their MSMS

analysis was done using MS-Seq module from Protein Prospector software of University of California, San Fransisco, USA (Clauser et al. 1999) and they corresponded to the sequences QKALNEINQF, TKKTKLTEE EKNRL from aS2-casein.

Conclusion The present study investigated the ACE inhibition, antioxidant activity, prolyl endopeptidase activity, antimicrobial activity of hydrolysates from bovine milk a-casein. The safety and efcacy of these peptides needs thorough study. This study identies these peptides as viable candidates for incorporation into functional foods and infant formulas perhaps helps in growth of the infants and also prevent them from infections.

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14
Intens. [a.u.]

Int J Pept Res Ther (2010) 16:715

1718.675

3000

Intens. [a.u.]

168.727

TKKTKLTEEEKNRL
1718.066 196.698

4000
1995.942

Intensity (AU)

2000

2108.056

3000

Intensity (AU)

50.470

b2

2000
111.790

1523.384

1881.812

2130.112

1000
1061.937 1203.072

1629.484

a2
392.612

257.628

b4 y13
436.604 1460.388 1618.804

1746.572

1000
2298.182 2336.259 2146.011 2188.899 2032.891 2242.750

b2

301.627

354.599

1023.950

1105.066 1152.123

850.863

1370.222

1265.273

1501.355

y4

0 00

0 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 200 400 600 800 1000 1200 1400 1600 1800

1095.660

1698.300

975.946

Intens. [a.u.]

m/Z

m/Z

2000 m/z

69.722

Q K ALNEINQF
479.334 225.454 496.390

4000

Intensity (AU)

3000
30.609 1202.000 610.364 1054.751 534.348

100.667

210.497

338.443

128.598

364.328

2000

182.565

165.510

381.413

424.418

y1
310.411

b4 y2 b2 b3 b6
627.469 666.381 707.409

461.320

778.536

y4

0 200 400 600 800 1000

910.530

960.121

1000

267.419

b7

y3

b8

m/Z

1200 m/z

Fig. 5 a Peptide mass ngerprinting of crude peptide fraction on MALDI-TOF. The two important peaks corresponding to the 1,204 and 1,718 Da were considered further as their sequences match a-casein. b The MALDI TOF MS/MS pattern of the 1,204 Da peptide. Following sequence interpretation and database searching the Acknowledgments Author S. Srinivas gratefully acknowledges the senior research fellowship during the course of the investigation from Council of Scientic and Industrial Research (CSIR), New Delhi, India.

1181.990

MS/MS pattern of the peptide was identied to peptide with QKALNEINQF from aS2-casein. c The MALDI TOF MS/MS pattern of the 1,718 Da peptide. Following sequence interpretation and database searching the MS/MS pattern was identied to peptide with TKKTKLTEEEKNRL from aS2-casein gastric and duodenal juice and the effects on selected microorganisms. Brit J Nutr 96:562569 Amiot J, Germain L, Turgeon S, Lemay M, Ory-Salam C, Francois FA, Auger A (2004) Peptides from milk protein hydrolysates to improve the growth of human keratinocytes in culture. Int Dairy J 14:619626 Ariyoshi Y (1993) Angiotensin converting enzyme inhibitors derived from food proteins. Tr Food Sci Tech 4:139144 Chiba H, Yoshikawa M (1989) Biologically functional peptides from milk proteins. In: Freeny RE, Whitaker JR (eds) Protein tailoring for food and medical uses. Marcel Dekker, New York, pp 123 153 Church CF, Swaisgood HE, Porter DH, Catignani GL (1983) Spectrophotometric assay using O-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J Dairy Sci 66:12191227 Clare DA, Swaisgood HE (2000) Bioactive milk peptides, a prospectus. J Dairy Sci 83:11871195

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