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Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Suwon, Gyeonggi, 441–706, Republic of Korea
Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.
Interest in probiotic bifidobacteria, which are gram-positive anaerobic bacteria found in human and animal intestinal tracts, has been increasing given their beneficial health effects including inhibition of pathogenic species, reduction of colon cancer risk, protective effects on immune function, regulation of gut microflora resistance to microbial infections and serum cholesterol reduction (
). Given their beneficial effects, strains such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, and Bifidobacterium bifidum are used as probiotic bacteria in the manufacture of milk products; for example, fermented milks, cheese, infant formulas, or food supplements (
Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk.
), similar to other LAB, including Lactococcus lactis, Lactobacillus rhamnosus, Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus helveticus, and Lactobacillus bulgaricus. They are auxotrophic for nitrogen sources (e.g., leucine and peptides;
). To obtain these nitrogen sources containing leucine, they use their proteolytic system as do other LAB. In fact, Bifidobacterium animalis ssp. lactis can hydrolyze milk protein to peptides using its proteolytic system (
). Some of these peptides have been reported to be bioactive. In general, bioactive peptides are generated from casein in different ways, such as (1) in vitro proteolysis and subsequent addition of the generated peptides to food, (2) in vivo gastrointestinal enzymatic digestion by microbial enzymes in the gastrointestinal tract of organisms, or (3) by lactic acid bacteria during fermentation (
). It is well known that milk proteins such as casein and whey proteins are precursor proteins that generate bioactive peptides released by the proteolytic system of LAB. Milk-based bioactive peptides released by LAB are well documented in the literature (
). For example, the VPP and IPP peptides from β-casein are best known for their angiotensin-converting enzyme (ACE) inhibition activity, and the fermented milks Calpis (Calpis Co. Ltd., Tokyo, Japan) and Evolus (Valio Ltd., Helsinki, Finland) containing the VPP and IPP peptides are commercially available (
). In addition, many peptides generated from casein by microbial fermentation are ACE inhibitory peptides, in contrast to other bioactive peptides (e.g., antioxidant peptides, opiates, antimutagens, immunomodulatory peptides, antimicrobial peptides, or peptides with mineral binding activity).
Among the various bioactive peptides, those with antioxidant properties are also important because increased levels of reactive oxygen species such as free radicals cause cancer, DNA damage, diabetes, cardiovascular diseases, allergies, and aging (
). Therefore, other antioxidant substances originating from plants or food, particularly milk protein from dairy products, may be provided to organisms as a supplement to prevent such damage.
However, production of bioactive peptides by Bifidobacterium is rarely documented in the literature, except for research on the hypocholesterolemic effect of casein hydrolysate after incubation with Bifidobacterium animalis ssp. lactis strain Bb12 and of yogurt containing Bifidobacterium pseudocatenulatum G4 (or Bifidobacterium longum BB536), reported by
, respectively. On the contrary, we can find no reports to date of other bioactive peptides including antioxidant peptides generated by Bifidobacterium strains. For this reason, the objective of this work was to investigate potential antioxidant peptides released from bovine casein after hydrolysis by Bifidobacterium longum KACC91563.
Materials and Methods
Materials
Chemical reagents including 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), gallic acid, Folin-Ciocalteu reagent, and the chromogenic substrate lysine-p-nitroanilic acid (Lys-pNA) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical grade.
Isolation and Retrieval of Bifidobacterium longum KACC91563
Matrix and standard preparation and identification of the isolate from infant feces were performed using a previously described method (
). After isolation of this strain, Bifidobacterium longum KACC91563 was grown in de Man, Rogosa, and Sharpe (MRS) broth (BD Biosciences, San Jose, CA) containing cysteine (0.05%, final concentration), and the cells (bacteria) were harvested by centrifugation (Beckman Coulter, Brea, CA) at 3,200 × g for 30 min at 4°C. The cells were stored in reconstituted skim milk (10% wt/vol) at −80°C.
Growth Conditions
Before the experiment, B. longum was grown overnight in skim milk at 37°C, according to the method previously described (
). Bifidobacterium longum (initial inoculum, 1%) was grown at 37°C in 50 mL of MRS broth supplemented with cysteine (final concentration, 1%).
Casein Hydrolysis
Bifidobacterium longum (initial inoculum, 1%) was grown at 37°C in 50 mL of MRS broth supplemented with cysteine (final concentration, 1%) to the exponential phase (pH 5.1). Then, the cells were harvested by centrifugation (Beckman Coulter) at 3,200 × g for 15 min at 20°C and washed twice with 0.05 M sodium phosphate buffer, pH 7.0, using the same centrifugation conditions. The cells were suspended in 5 mL of the same buffer and mixed with 20 mL of 0.1% (wt/vol) bovine casein (Sigma-Aldrich) solution as a substrate. The cells were then incubated at 37°C for 0, 4, 24, and 48 h, and the supernatant fractions were immediately collected after centrifugation at 13,200 × g for 10 min at 4°C and filtered (0.45-μm filter). The hydrolysates obtained were stored at −20°C before their use in the following experiments.
Peptidase Activity
To verify cell surface peptidase and cell lysis activity of B. longum, a peptidase activity assay was performed using the chromogenic substrate Lys-pNA (
). The substrate was prepared at a concentration of 2 mM in 0.05 M sodium phosphate buffer, pH 7.0. Bifidobacterium longum grown to the exponential phase (pH 5.1) was harvested by centrifugation at 3,200 × g for 5 min at 20°C. The cell pellet was washed 2 times with 0.05 M sodium phosphate buffer, pH 7.0, and then resuspended in the same buffer. One hundred microliters of cell suspension was then mixed with 1 mL of Lys-pNA solution and incubated for 1 h at 37°C. The reaction mixture was centrifuged at 13,200 × g at 4°C for 10 min to pellet the cells, and the absorbance of the supernatant was determined at 410 nm using a SpectraMax spectrophotometer (Molecular Devices, Sunnyvale, CA).
Electrophoresis
Sodium dodecyl sulfate-PAGE was performed as previously described (
) using a 5% stacking gel and 15% separating gel. Twenty microliters (about 10 μg of hydrolysate) was loaded into each well along with broad-range standard molecular mass markers (6, 17, 26, 34, 43, 55, 70, 95, 130, and 170 kDa). After migration, the gel was stained in 0.1% Coomassie Blue R-250 solution containing 2% (wt/vol) TCA and 50% (vol/vol) ethanol. The gel was destained using a mixture of 50% methanol, 10% acetic acid, and 40% water.
Reverse-Phase HPLC
To confirm the hydrolysis of bovine casein, the hydrolysates were separated on a Zorbax 300SB-C18 analytical column (3.0 × 150 mm, diameter 3.5 μm, Agilent, Santa Clara, CA) connected to an HPLC system (Jasco, Oklahoma City, OK). Fifty microliters of sample were injected into the column and eluted at 30°C using a linear gradient (5 to 50%) over 90 min. The flow rate was 0.25 mL/min, and peptides were detected by UV absorption at 215 nm.
Antioxidant Activity: Radical Scavenging Assay
Free radical scavenging activity of the casein hydrolysate was determined using ABTS radical cation (ABTS+•) according to previously described method with minor modifications (
). To generate the ABTS+• radical, ABTS and potassium persulfate were prepared at final concentrations of 7 and 2.45 mM, respectively, and incubated for 16 h in the dark at 30°C. Using a spectrophotometer (Molecular Devices), the ABTS+• radical was adjusted to an absorbance of 0.70 ± 0.02 at 735 nm by diluting with distilled water. Fifty microliters of casein hydrolysate was mixed with 950 μL of the ABTS+• radical solution. The decrease of the absorbance was monitored at 735 nm after 30 min at 30°C. All assays were carried out in triplicate, and the values represent the means. A standard curve was prepared using various concentrations (1, 2, 20, 40, 60, 80, 100, and 120 μM) of gallic acid. For standard curve calculation, the percentage antioxidant activity was calculated as follows: Scavenging activity (%) = [(Acontrol − Asample)/Acontrol] × 100, where Acontrol represents the initial ABTS absorbance. The radical scavenging activity of the casein hydrolysates was calculated from the equation of the standard curve based on gallic acid and expressed as micromoles of gallic acid equivalent (GE).
Antioxidant Activity: Determination of Total Phenolic Contents
Total phenolic content (TPC) measurement was followed by the previously described method (
) with minor modifications. For this purpose, a volume of 60 µL of sample was mixed with 60 µL of the Folin-Ciocalteu solution, which was prepared by mixing 1 volume of Folin-Ciocalteu reagent with 2 volumes of distilled water. Finally, 60 µL of 10% sodium carbonate (Na2CO3) solution was added in this mixture. After incubation of the mixture at room temperature for 1 h in the dark, absorbance at 700 nm was measured using a spectrophotometer (Molecular Devices). All assays were carried out in triplicate and the values represent the means. A standard curve was prepared using various concentrations (1, 2, 20, 40, 60, 80, 100, and 120 μM) of gallic acid. The TPC of the casein hydrolysates was calculated from the equation of the standard curve based on gallic acid and expressed as micromoles of GE.
Fractionation of Hydrolysates
Fractions with molecular weight cutoffs of 10 and 3 kDa were separated using an ultrafiltration membrane system (Millipore, Billerica, MA).
Mass Spectrometry Analysis
Liquid chromatography tandem MS (LC-MS/MS) experiments were carried out using an integrated system consisting of autoswitching nano pump, autosampler (Tempo nano LC system, MDS Sciex, Toronto, Ontario, Canada) and a hybrid quadrupole-time-of-flight (TOF) tandem mass spectrometer (QStar Elite, Applied Biosystems, Foster City, CA) equipped with a nano-electrospray ionization (ESI) source and fitted with a fused-silica emitter tip (New Objective, Woburn, MA). This experiment was performed at National Instrumentation Center for Environmental Management (NICEM) of Seoul National University in Korea. Fractions (2 μL) were injected into LC-nano ESI-MS/MS system. Solvent A consisted of water/acetonitrile (98:2, vol/vol) with 0.1% formic acid for the high aqueous mobile phase. Samples were first trapped on a Zorbax 300SB-C18 trap column (300-μm i.d. × 5 mm, 5-μm particle size, 100 Å pore size, part number 5065–9913; Agilent Technologies, Santa Clara, CA) and washed for 6 min with 98% solvent A [water/ACN (98:2, vol/vol), 0.1% formic acid] and 2% solvent B [water/ACN (2:98, vol/vol), 0.1% formic acid] at a flow rate of 5 μL/min, and then separated on a Zorbax 300SB-C18 capillary column (75-μm i.d. × 150 mm, 3.5-μm, 100 Å pore size, part number 5065–9911; Agilent Technologies) at a flow rate of 300 nL/min. The LC gradient was run at 2 to 35% solvent B over 30 min, and then from 35 to 90% over 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min. Resulting peptides were electrosprayed through a coated silica tip (FS360-20-10-N20-C12, PicoTip emitter, New Objective) at an ion spray voltage of 2,000 eV. Mass data were acquired automatically using Analyst QS 2.0 software (Applied Biosystems). The range of m/z was 200 to 2,000.
Results and Discussion
Production of Peptides from Casein Hydrolysis of Bifidobacterium longum
Bifidobacterium longum was grown to exponential phase in MRS broth containing cysteine. The cells were retrieved and incubated with bovine casein in 0.05 M sodium phosphate buffer, pH 7.0. Casein hydrolysates were obtained after 0, 4, 24, and 48 h of hydrolysis by centrifugation and filtration (0.45 μm). Bovine casein was solubilized in 0.05 M sodium phosphate buffer at pH 7. This pH is similar to that of milk (6.6–6.8), indicating the similar environment (
The casein hydrolysates were analyzed by SDS-PAGE to verify the hydrolysis patterns after the above-mentioned incubation periods. Figure 1 shows the hydrolysis of bovine casein by B. longum after 24 and 48 h of incubation, in particular, of β-casein compared with αS1-casein and αS2-casein. Similar observations have been reported in several studies on LAB in the literature, including Lc. lactis, Lb. rhamnosus, Strep. thermophilus, Lb. lactis, Lb. helveticus, and Lb. bulgaricus (
). Thus, in the case of B. longum, as with other LAB, β-casein is preferentially degraded compared with αS1-casein and αS2-casein. The different accessibilities may be explained by structural differences between individual caseins. Compared with other caseins, β-casein is more unstructured and more accessible to cleavage, and is therefore hydrolyzed to a greater extent (
Figure 1Electrophoretic patterns of bovine casein hydrolyzed by Bifidobacterium longum KACC91563. Twenty microliters (about 10 μg of hydrolysate) was loaded in each well. M denotes protein molecular weight markers. Cells were grown in de Man, Rogosa, and Sharpe broth until the pH reached 5.1 and then were collected after centrifugation. Caseins were solubilized at 0.1% (wt/vol) in 0.05 M Na phosphate buffer, pH 7.0. One volume of cell suspension of B. longum was incubated with 4 volumes of caseins at 37°C and the hydrolysate used as a sample was recovered at each incubation time: (a) 0 h, (b) 4 h, (c) 24 h, and (d) 48 h.
This observation of casein degradation was confirmed by HPLC profiles. As shown in Figure 2, the number of peaks increased with incubation time, especially after hydrolysis for 24 and 48 h, in contrast to the minimal casein degradation seen at elution times under about 40 min at 0 h of incubation. This indicates that the casein is well degraded by B. longum in the present study, although no protease activity was detected for B. longum using the chromogenic substrate (Suc-Ala-Ala-Pro-Phe-pNa) and zymography (data not shown). This has also been proven by
, who reported the absence of protease activity of B. animalis ssp. lactis in whole cells, the cell wall-bound fraction, and cell extracts. In some strains such as B. longum DJO10A (accession number YP_001955201.1) or B. longum ssp. infantis ATCC 15697 (accession number YP_002322571.1), a subtilisin-like serine protease is present, but there are no reports of the genetic and biochemical characterization and identification for this protease, similar to the cell envelope protease (CEP) of LAB. In fact, some LAB use their CEP to hydrolyze milk protein for growth (
Figure 2Reverse phase-HPLC analysis of bovine casein (0.1%, wt/vol) hydrolysis by fermentation of Bifidobacterium longum KACC9156. Fifty microliters of sample was injected onto the column and eluted at 30°C by using a linear gradient (5 to 50%) of solvent B (0.1% trifluoroacetic acid in acetonitrile) within 90 min. The flow rate was 0.25 mL/min, and peptides were detected by UV absorption at 215 nm.
On the other hand, whole cells in the B. animalis strain can degrade casein after 24 h of incubation; this could be a result of cell lysis, causing degradation of casein by enzymes in the intracellular fraction, such as peptidase O (pep O) with 67.4% identity in B. longum (
). However, we observed no peptidase activity using Lys-pNA in cell suspensions without casein after 0, 4, 24, and 48 h of incubation in the present study, which indicates the absence of cell lysis. Therefore, the degradation of casein by B. longum was not due to intracellular peptidases released by cell lysis. However, peptidase activity using Lys-pNA was detected in the whole cell fraction [absorbance at 410 nm (A410) = 0.921, which corresponds to about 38.4 nmol/min per mL of pNA released, calculated by Lambert-Beer law’s equation]. This means that casein could be hydrolyzed by potential peptidases linked to the cell wall or another proteolytic system. Further study is needed to identify and characterize this peptidase.
Investigation of Antioxidant Activity in Hydrolyzed Fractions Using ABTS Radical Scavenging Capacity
To investigate the free radical scavenging properties of casein hydrolysates by B. longum according to incubation times, the ABTS method, which is widely used to investigate free radical scavenging properties for antioxidant activity, was adapted from a previously described method (
). In the present study, none of the casein hydrolysate fractions were detected with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, another free radical scavenging activity assay (data not shown). This could be due to the nature of compounds in the sample.
reported that the ABTS radical is soluble in aqueous and organic media, whereas the DPPH radical dissolves only in organic media. Therefore, in this study, the hydrolysate fractions were analyzed using the ABTS radical scavenging method.
Various concentrations of gallic acid, a strong antioxidant molecule, were used to generate the standard curve. The reaction mixture (gallic acid and ABTS cation radical) was incubated at 30°C for 30 min. The linear standard curve with a range from 0 to 120 μM gallic acid is presented in Figure 3A. From the equation of the standard curve, the IC50 value (the concentration that inhibits the activity by 50%) was 56 μM gallic acid.
Figure 3Relationship between concentration and ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] free radical scavenging capacity (A) and total phenolic contents (B) of gallic acid as a standard. Data are expressed as the average value of triplicates. The equation for this curve is y = 0.8106x + 4.377, with an R2 value of 0.99 for ABTS free radical scavenging capacity and y = 0.0182 × − 0.0039; R2 = 0.99 for total phenolic contents.
The free radical scavenging properties of casein hydrolysates following different incubation periods are presented in Table 1. Antioxidant properties improved with increasing duration of hydrolysis with B. longum. The 24- and 48-h hydrolysates exhibited higher antioxidant capacities than did the other hydrolysates. Their GE values deduced from the standard curve equation of gallic acid were 2,045.5 and 1,629.3 μM, respectively (Table 1). A blank sample (casein solution after 24-h incubation without B. longum cells) was measured as a control for casein autolysis and showed a GE value similar to that seen at 0 h. Thus, it appears that casein autolysis does not result in antioxidant activity at 4, 24, or 48 h. The different GE values may be due to more extensive hydrolysis at 48 h than at 24 h. Consequently, the smaller peptides generated after 48 h of hydrolysis may lack antioxidant activity.
Table 1Antioxidant activities in gallic acid equivalents (GE, μM) of casein hydrolysates after incubation with Bifidobacterium longum KACC91563
ABTS=2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); gallic acid equivalents were calculated using the standard curve equation after triplicate measurement of absorbance (at 735nm for ABTS and at 700nm for TPC).
Incubation time (h)
0
4
24
48
ABTS free radical scavenging capacity
120.1
140.3
2,045.5
1,629.3
Total phenolic content (TPC)
6.5
43.2
52.4
20.2
1 ABTS = 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); gallic acid equivalents were calculated using the standard curve equation after triplicate measurement of absorbance (at 735 nm for ABTS and at 700 nm for TPC).
The GE values at 0 and 4 h of hydrolysis were 120.1 and 140.3 μM, respectively (Table 1), which corresponded to 5.8 and 6.8% (0 and 4 h vs. 24 h) and 7.3 and 8.6% (0 and 4 h vs. 48 h) of the later values, respectively. Thus, the 24-h and 48-h hydrolysates may contain antioxidant peptides (or antioxidant substances) from bovine casein.
Investigation of TPC in Hydrolyzed Fractions
Phenolic content analysis was performed to detect phenolic amino acids in the peptides and to confirm antioxidant activity. Gallic acid was used to generate the standard curve. The linear standard curve with a range from 0 to 100 μM gallic acid is presented in Figure 3B. The GE values of TPC in the <3 kDa fraction correspond to 6.5, 43.2, 52.4, and 20.2 μM for 0, 4, 24, and 48 h, respectively (Table 1), showing low GE val ues compared with those seen with ABTS scavenging activity. Unlike the results of ABTS radical scavenging activity, the hydrolysate after 4 h of incubation had a high GE value (43.5 μM) compared with that at 48 h (20.2 μM). Regardless of the antioxidant activity measurement method, the highest GE value was seen for the hydrolysate after 24 h incubation. This fraction had a relatively low content of peptides with phenol groups (e.g., tyrosine) compared with other antioxidant peptides detected by ABTS scavenging activity.
Antioxidant Properties in Fractions Separated According to Molecular Weight by Ultrafiltration
For further analysis, the 24-h hydrolysate was fractionated according to molecular weight using ultrafiltration. Using cut-off values of 10 and 3 kDa, the 24-h hydrolysate was separated into 3 fractions (>10 kDa, 3–10 kDa, and <3 kDa). The <3 kDa fraction exhibited the highest free radical scavenging capacity after 10-fold dilution (80.3%), corresponding to 936.7 μM GE (Table 2). The capacities of the >10 kDa and 3–10 kDa fractions were 38.5 and 32.1%, respectively, which corresponded to 42.1 and 34.2 μM GE, respectively. Thus, potential antioxidant peptides derived from bovine casein hydrolysis with B. longum may be present in the <3 kDa fraction, given that most milk protein-derived bioactive peptides with antioxidant properties reported in the literature are small (<10 AA). Thus, this fraction was used for the identification of peptides using LC-ESI-MS/MS analysis.
Table 2Antioxidant activities of casein hydrolysate fractions obtained after 24 h of hydrolysis by Bifidobacterium longum KACC91563
Identification of Peptides Generated from Bovine Casein in the <3 kDa Fraction
The peptides generated from bovine casein were further identified by LC-ESI-quantitative time-of-flight MS analysis. In the <3 kDa fraction, the peptides were generated to a greater extent from β-casein than from other caseins (α-casein and κ-casein), consistent with results of the SDS-PAGE and reversed phase-HPLC profiles. Nineteen peptides were generated from β-casein, but only 12 were generated from αS1-casein and 2 from κ-casein (Table 3).
Table 3Casein-derived peptides identified by liquid chromatography-electrospray ionization-tandem mass spectrometry in the <3 kDa fraction of the 24-h hydrolysate obtained by fermentation of Bifidobacterium longum KACC91563
). To date, 6 types of CEP in LAB have been reported, including PrtP for Lc. lactis, PrtR for Lb. rhamnosus, PrtS for Strep. thermophilus, PrtL for Lb. lactis, PrtH for Lb. helveticus, and PrtB for Lb. bulgaricus; these facilitate bacterial growth themselves and produce peptides (
). As mentioned above, in the case of B. longum, no CEP activity was detected in the cell wall using zymography and using the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA (Sigma). Similar to B. longum in the present study, the Strep. thermophilus CNRZ1066 strain without the gene encoding CEP did not hydrolyze casein. This strain also had no intracellular peptidase activity upon cell lysis and no other cell wall-bound peptidase activity (
). In contrast, in the present work, peptidase activity was detected in the whole-cell fraction without cell lysis during incubation, which was confirmed using Lys-pNA. Thus, in B. longum, casein could be hydrolyzed by this potential CEP or another proteolytic system.
The hydrophilic region at the N-terminal region on β-casein was resistant to hydrolysis by B. longum (Figure 4). This observation was also made in previous studies showing that the hydrophobic region (especially the C-terminal region) was more accessible to the CEP of Strep. thermophilus (
Figure 4Cleavage region of peptide bonds on bovine β-, αS1-, and κ-caseins hydrolyzed after fermentation with Bifidobacterium longum KACC9156. The arrows indicate cleaved peptide bonds.
In the case of αS1-casein, 12 peptides were generated by the fermentation of B. longum. The N-terminal region was more accessible to hydrolysis by this strain (Figure 4). The αS1-casein structure is composed of 4 parts (
): (1) hydrophilic region (f1–12), (2) hydrophobic region (f13–40), (3) hydrophilic region (to residue 100), (4) hydrophobic region (f100–199). In the present study, αS1-casein was degraded to a greater extent in the hydrophobic region (f8–35) than in the hydrophilic region, and only 1 peptide was generated in the C-terminal hydrophobic region (f179–199), indicating high resistance in the region of residues 40–175 and residues 181–199. This was similar to the results of
, who reported that the region of residues 1–40 is most susceptible to hydrolysis, whereas the region 41–91 is very resistant to hydrolysis. This observation on the accessibility of each region is not consistent with that of β-casein in the present study.
Only 2 peptides were generated from the N-terminal region of κ-casein in the present work. No peptide was generated from the glycomacropeptide (f106–169) region at the C-terminus. Glycan chains may protect against hydrolysis in this region conferring hydrophilic properties and negative charge (increase in electrostatic repulsions;
Zahraa N. 2010. Le peptide κ-CN(f106–109) du lait: Propriétés nutritionnelles, biologiques et techno-fonctionnelles. Mémoire de M2, UHP Nancy 1. Université Henri Poincaré (UHP) Nancy 1, Nancy, France.
No peptides were identified from αS2-casein in this fraction. αS2-Casein could be more resistant to hydrolysis by B. longum due to the formation of a tetrameric form of αS2-casein in phosphate buffer (pH 8.1), providing protection in some regions (
). Investigation of the accessibility of regions containing protein structure seems to be an appropriate method to explain susceptibility to hydrolysis (
Potential Antioxidant Peptide in the <3 kDa Fraction
In previous studies, antioxidant peptides have been investigated following the hydrolysis of individual caseins (Table 4). Milk proteins and fermented milk are well-known, food-based sources of natural antioxidants in the form of antioxidant peptides (
As shown in Table 4, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, among the peptides generated from β-casein identified through the analysis in this work exhibited antioxidant properties. These peptides contain the fragment β-casein (f185–190), VLPVPQ, which has antioxidant activity. This is consistent with studies by
, who reported that the peptide β-casein (f181–190), SQSKVLPVPQ, may contain a potential antioxidant property, given that this peptide contains the fragment β-casein (f185–190), VLPVPQ, which was previously identified from Spanish commercial fermented milk with Lactobacillus helveticus and Saccharomyces cerevisiae.
reported that the peptide VLSLSQSKVLPVPQKAVPYPQRDMPIQA, derived from milk protein after trypsin treatment and containing the pep tide β-casein (f177–183), AVPYPQR, has antioxidant activity on the basis of its DPPH scavenging activity. Thus, antioxidant peptide(s) could be generated from β-casein by B. longum.
In addition, the peptide β-casein (f98–105), VKEAMAPK, has been reported to have antioxidant properties. Recently, PrtS of Strep. thermophilus 4F44 has been shown to generate this fragment (
). In a synthetic peptide β-casein (f98–105), the antioxidant properties were determined through enzymatically and chemically induced oxidation of the linoleic acid oxidation system, hemoglobin-catalyzed oxidation of linoleic acid, hydroperoxide oxidation, DPPH assay, and measurement of Fe2+ chelating activity (
For αS1-casein, the peptide αS1-casein (f144–149), YFYPEL, showed strong superoxide anion radical scavenging activity. This peptide was isolated from pepsic hydrolysate and exhibited superoxide anion scavenging activity and DPPH radical and hydroxyl radical scavenging activities (
). The fragment PYVRYL from ovine αS2-casein has antioxidant properties and corresponds to the αS2-casein (f202–207) fragment (PYVRYL) from bovine αS2-casein generated after hydrolysis by the cell envelope protease PrtS of Strep. thermophilus PB385 (
However, no antioxidant peptides were detected from αS1- and κ-casein in the present results. This could be explained by differences in protein degradation with or without proteases during incubation between LAB and Bifidobacterium.
Conclusions
In the present study, antioxidant activity in casein-derived peptides obtained from fermentation with B. longum was detected using ABTS free radical scavenging activity and total phenolic peptide measurements. Two peptides having antioxidant activity were identified through MS analysis. Based on these results, further study of the <3 kDa fraction of each casein and fermented milk is needed to investigate and identify novel peptides with antioxidant properties and to screen bioactive peptides generated by B. longum that have other functions, such as antihypertensive, antimutagenic, and immunomodulant peptides.
Acknowledgments
This study was sponsored by National Institute of Animal Science (NIAS) research project (PJ0085852013) of Rural Development Administration of Korea (RDA). The author Oun Ki Chang participated in this project as a postdoctoral researcher. The authors acknowledge J. Lee and researcher H.-J. Choi (both from the National Instrumentation Center for Environmental Management, Seoul National University, Seoul, Korea) for MS analysis.
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