Identification of Antihypertensive Peptides Derived from Low Molecular Weight Casein Hydrolysates Generated during Fermentation by Bifidobacterium longum KACC 91563
Identification of Antihypertensive Peptides Derived from Low Molecular Weight Casein Hydrolysates Generated during Fermentation by Bifidobacterium longum KACC 91563
Food Science of Animal Resources. 2015. Dec, 35(6): 738-747
Copyright © 2015, Korean Society for Food Science of Animal Resources
This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : May 11, 2015
  • Accepted : August 03, 2015
  • Published : December 31, 2015
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About the Authors
Go Eun, Ha
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea
Oun Ki, Chang
Imported Food Analysis Division, Ministry of Food and Drug Safety, Gwangju 61012, Korea
Su-Mi, Jo
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea
Gi-Sung, Han
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea
Beom-Young, Park
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea
Jun-Sang, Ham
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea
Seok-Geun, Jeong
Animal Products Research and Development Division, National Institute of Animal Science, RDA, Jeonju 55365, Korea

Angiotensin-converting enzyme (ACE) inhibitory activity was evaluated for the low-molecular-weight fraction (<3 kDa) obtained from milk fermentation by Bifidobacterium longum KACC91563. The ACE inhibitory activity in this fraction was 62.3%. The peptides generated from the <3 kDa fraction were identified by liquid chromatography-electrospray ionization quantitative time-of-flight mass spectrometry analysis. Of the 28 peptides identified, 11 and 16 were identified as β-casein (CN) and α s1 -CN, respectively. One peptide was identified as κ-CN. Three peptides, YQEPVLGPVRGPFPIIV, QEPVLGPVRGPFPIIV, and GPVRGPFPIIV, from β-CN corresponded to known antihypertensive peptides. We also found 15 peptides that were identified as potential antihypertensive peptides because they included a known antihypertensive peptide fragment. These peptides were as follows: RELEELNVPGEIVE (f1-14), YQEPVLGPVRGPFP (f193-206), EPVLGPVRGPFPIIV (f195-206), PVLGPVRGPFPIIV (f196-206), VLGPVRGPFPIIV (f197-206), and LGPVRGPFPIIV (f198-206) for β-CN; and APSFSDIPNPIGSENSEKTTMPLW (f176-199), SFSDIPNPIGSENSEKT- TMPLW (f178-199), FSDIPNPIGSENSEKTTMPLW (f179-199), SDIPNPIGSENSEKTTMPLW (f180-199), DIPNPIGSENSEKTTMPLW (f181-199), IPNPIGSENSEKTTMPLW (f182-199), PIGSENSEKTTMPLW (f185-199), IGSENSEKTTMPLW (f186-199), and SENSEKTTMPLW (f188-199) for α s1 -CN. From these results, B. longum could be used as a starter culture in combination with other lactic acid bacteria in the dairy industry, and/or these peptides could be used in functional food manufacturing as additives for the development of a product with beneficial effects for human health.
Probiotic bifidobacteria such as Bifidobacterium longum ( B. longum ), Bifidobacterium breve , Bifidobacterium animalis , and Bifidobacterium bifidum are commonly used as starter cultures in the dairy industry e.g., fermented milk, cheese and infant formulas ( Chang , 2013 ; Davidson , 2000 ; Martín-Diana , 2003 ; McBrearty , 2001 ; Saavedra , 2004 ) because of their beneficial effects for human health. The various beneficial effects include the pathogenic species inhibition, diminution of colon cancer risk, immune response for protection effect on their function, regulation of gut microflora ( Arunachalam, 1999 ; Chang , 2013 ; Collins and Gibson, 1999 ; Leahy , 2005 ).
As lactic acid bacteria (LAB) including Lactococcus lactis ( L. lactis ), Lactobacillus rhamnosus ( Lb. rhamnosus ), Streptococcus thermophilus ( St. thermophilus ), Lactobacillus lactis ( Lb. lactis ), Lactobacillus helveticus ( Lb. helveticus ), and Lactobacillus subsp. bulgaricus ( Lb. bulgaricus ), bifidobacteria strains for their growth also use milk protein as a nitrogen source, and their proteolytic system can produce peptides in milk ( Chang , 2013 ; Janer , 2005 ). The proteolytic system of LAB as St. thermophilus , Lb. rhamnosus , L. lactis , Lb. bulgaricus , Lb. helveticus is composed of 3 steps which contain the cell envelope protease (CEP), a transporter of oligopeptides and small peptides, and the various intracellular peptidases ( Genay , 2009 ; Gilbert , 1996 ; Miclo , 2012 ; Pastar , 2003 ; Sadat-Mekmene , 2011b ; Siezen, 1999 ).
The proteolytic system of bifidobacteria is not well known. Some studies have shown that peptidases of the genus Bifidobacterium can hydrolyze milk protein directly, including those of Bifidobacterium animalis subsp. lactis ( Janer , 2005 ), Bifidobacterium longum Bl 536 ( Donkor , 2007 ), and B. longum KACC 91563 ( Chang , 2013 ) generating the biological peptides. Hydrolysis of milk protein by microorganisms during fermentation can generate various peptides, including biologically active peptides, e.g., angiotensin I-converting enzyme (ACE) inhibitory peptides, antioxidant peptides, opiates, antimutagens, immunomodulatory peptides, antimicrobial peptides, or peptides with mineral binding activity. The bioactive peptides produced by the activity of proteases or peptidases of microorganisms are well documented in the literature ( Korhonen, 2009 ). The most studied commercial fermented milk products are “Calpis” and “Evolus”, including two ACE-inhibitory tripeptides, VPP and IPP generated from β-casein by fermentation with Lb. helveticus including Saccharomyces cerevisiae for Calpis and Lb. helveticus for Evolus have been commercialized ( Korhonen, 2009 ; Seppo , 2002 ; Takano, 2002 ).
In the case of bifidobacteria, the antioxidative peptides produced by B. longum were reported by Chang (2013) . Another study showed that the bioactive peptides of B. longum Bl 536 presented in vitro ACE-inhibitory activity, indicating that these are potential hypotensive peptides ( Donkor , 2007 ); however, the peptides were not identified. Inhibition of ACE activity is regarded as an important component for the treatment of patients with hypertension because ACE leads to an increase in blood pressure by conversion of angiotensin I to angiogestin II or by bradykinin hydrolysis ( Gobbetti , 2000 ; Hayes , 2007a ; Miguel , 2009 ; Petrillo and Ondetti, 1982 ). Recently, some novel ACE-inhibitory peptides, including LVYPFP, were identified in Bifidobacterium bifidum ( Gonzalez-Gonzalez , 2013 ). However, no hypotensive peptides have been identified in B. longum to date. Thus, we sought to investigate the antihypertensive activity and identify the peptides released from casein (CN) during fermentation of milk by B. longum KACC 91563.
Material and Methods
- Materials
Chemical reagents, including hippuryl-histydil-leucine(HHL), captopril, ACE (2 mU; EC, 5.1 U mg −1 ), and lung acetone powder from rabbit were purchased from Sigma Aldrich (USA), and all other chemicals used were of analytical grade.
- Preparation and growth ofBifidobacterium longumKACC91563
B. longum KACC91563 was isolated from infant feces in Korea. Identification of this strain was performed according to previously described methods ( Chang , 2013 ; Ham , 2011 ). After isolation, B. longum was grown according to the conditions described by Ruiz (2009) . B. longum KACC91563 was grown in de Man, Rogosa, and Sharpe (MRS) broth (BD Biosciences, USA) containing cysteine (0.05% final concentration), and the cells (bacteria) were harvested by centrifugation (Beckman Coulter, USA) at 3,200 g for 30 min at 4℃. The cells were incubated and stored in reconstituted skim milk (10% w/v) at 80℃.
- Milk fermentation
To obtain the fermentate fraction, fermentation was performed in skimmed milk for 24 h by inoculation of 1% B. longum after preculture at 37℃. The fermentate was retrieved by centrifugation at 3,200 g for 15 min at 4℃ when the fermentation was complete. The obtained fractions were separated using an ultrafiltration membrane system (Millipore, USA) with a molecular weight cut-off of 3 kDa.
- Measurement of ACE inhibitory activity
ACE inhibitory activity was measured using a spectrophotometric assay as previously described ( Cushman and Cheung, 1971 ) with slight modifications. First, crude ACE was prepared and extracted from 1 g of rabbit lung acetone powder (L0756, Sigma Aldrich) by gentle mixing of 40 mL of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl over 24 h. The supernatant containing ACE was obtained by centrifugation a 3,200 g for 40 min at 4℃, and was used for ACE inhibitory activity. A 50 μL sample was added to 100 μL of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl, and 50 μL of crude ACE was obtained. The reaction mixture was pre-incubated at 37℃ for 5 min. Fifty microliters of 12.5 mM HHL (H-1635, Sigma Aldrich) solubilized in 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl was added to the reaction mixture and left to stand for 30 min at 37℃ after vortexing. To stop the enzyme reaction, 250 μL of 1 N HCl was added. Next, 1.5 mL of ethyl acetate was added to this reaction mixture and vortexed for 15 s. One milliliter of supernatant was retrieved by centrifugation at 3,000 g for 5 min and evaporated using Concentrator Plus (Eppendorf, Germany) at 60℃ for 30 min. The product was resupended in 1 mL of distilled water and its absorbance was measured at 228 nm using a spectrophotometer (Molecular Devices, USA). All assays were carried out in triplicate and the values represent the average and standard errors. Captopril (C-4042, Sigma Aldrich) was used as a positive control. The ACE inhibitory activity was calculated as follows: ACE inhibition (%) = [(C − B) − (S − B)] × 100/(C − B), where S is the absorbance of the ACE, ACE-inhibitory sample, and HHL; B is the absorbance of ACE and sodium borate buffer (pH 8.3) without HHL; and C is the absorbance of ACE, sodium borate buffer (pH 8.3), and HHL.
- Identification of peptides by mass spectrometry
Liquid chromatography-electrospray ionization-quantitative time-of-flight tandem mass spectrometry experiments (LC-ESI-TOF-MS/MS) were performed at the National Instrumentation Center for Environmental Management (NICEM) of Seoul National University in Korea, according to the method described by Chang (2013) .
MS analysis experiments were carried out using an integrated system consisting of an auto-switching nano pump, autosampler (Tempo TM nano LC system; MDS SCIEX, Canada), and a hybrid quadrupole-time-of-flight (TOF) mass spectrometer (QStar Elite; Applied Biosystems, USA) fitted with a fused silica emitter tip (New Objective, USA). For ionization, the nano-electrospray ionization (ESI) was applied. Two μL fractions were injected into the LC-nano ESI-MS/MS system.
Samples were first trapped on a ZORBAX 300SB-C18 trap column (300-μm i.d × 5 mm, 5-μm particle size, 100 pore size, Agilent Technologies, part number 5065-9913) and washed for 6 min with gradient with 98% solvent A and 2% solvent B at a flow rate of 5 μL/min. The solvent A and B consisted in [water/acetonitrile (98:2, v/v), 0.1% formic acid] and [Water/acetonitrile (2:98, v/v), 0.1% formic acid]. Separation was carried out on a ZORBAX 300SB-C18 capillary column (75-μm i.d × 150 mm, 3.5-μm particle size, 100 pore size, part number 5065-9911) at a flow rate of 300 nL/min with gradient at 2% to 35% solvent B over 30 min, then from 35% to 90% over 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min. Electrospray through a coated silica tip (FS360-20-10-N20-C12, PicoTip emitter, New Objective) was performed at an ion spray voltage of 2,000 eV. Peptides were analyzed automatically using Analyst QS 2.0 software (Applied Biosystems, USA). The range of m/z values was 200-2000.
Results and Discussion
- Determination ofin vitroACE inhibitory activity in low molecular weight fermentate
Two fractions were prepared to evaluate the ACE inhibitory activity. One was the fraction obtained after B. longum KACC9156 fermentation in skimmed milk for 24 h. Fermentates were fractionated at a molecular weight cutoff of 3 kDa using a centrifugal ultrafiltration membrane system. This cutoff value was chosen because Gonzalez-Gonzalez (2013) demonstrated that the <3 kDa fraction of B. bifidum MF 20/5-fermented milk showed higher ACE inhibitory activity than that of the >3-kDa fraction. Similarly, Miguel (2009) showed that the 50% inhibitory concentration of ACE was higher (5.5 μg/mL) in the <3 kDa fraction than in the > 3 kDa fractions of the bovine CN hydrolysate and whole hydrolysate without molecular weight fractionation. Confirming these results, in the present study, the ACE inhibitory activity determined from the fermentate obtained from milk fermentation with B. longum was 62.3% ( Table 1 ), which was higher than that of the CN hydrolysate (28.3% ACE inhibitory activity) obtained from a 0.1% CN solution in 0.05 M sodium phosphate buffer (pH 7.0) at the same incubation time (data not shown). This ACE inhibitory activity was similar to that reported previously (63.7%) in milk fermented with B. longum B1 536 ( Donkor 2007 ). During fermentation, the casein could be degraded by the protolytic system of B. longum which was detected on peptidase activity of their cell surface ( Chang , 2013 ). However, the cell envelop protease of B. longum which is the first step of casein hydrolysis was not found. From this reason, the ACE inhibitory activity in this work could be resulted from the potential cell wall peptidase.
ACE-inhibitory activities in the low-molecular-weight fraction (<3 kDa) of the fermentate obtained after incubation of milk byBifidobacterium longumKACC 91563
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All assays were carried out in triplicate.
Using LC-ESI-MS/MS, the further study was proceeded to identify the peptides and to search the antihypertensive peptides generated by fermentation with B. longum KACC 9156 in milk.
- Peptides generated from milk casein during fermentation byBifidobacterium longumKACC9156
The peptides generated from fermentates were identified by LC-ESI-TOF-MS/MS. As shown in Table 2 , the peptides in the fermentate were identified to have been generated from CN. The results also indicated that CN was the preferential substrate of B. longum in spite of the presence of whey protein in skim milk, similar to a strain of St. thermophilus ( Chang , 2014 ). A total of 28 peptides were generated, corresponding to 11 β-CN, 16 α s1 -CN, and 1 κ-CN peptide. This observation was slightly different from that reported by Chang (2013) , who identified 33 peptides (19 β-CN, 12 α s1 -CN, and 2 κ-CN) in bovine CN hydrolysates obtained from a 0.1% CN solution with the same strain of B. longum . This difference could be explained by a difference in the accessibility of peptides for hydrolysis by B. longum or in a structural difference of milk protein in the different matrices used, i.e., milk versus buffer.
Casein-derived peptides identified by liquid chromatography electrospray ionization time-of-flight tandem mass spectrometry in the <3 kDa fraction of the 24 h fermentates obtained from fermentation of milk byBifidobacterium longumKACC91563
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CN: casein, m/z = mass to charge ratio, where z = number of positively charged ions.
Nonetheless, the pattern of CN hydrolysis determined in the present study was similar to that reported by Chang (2013) . In the case of β-CN peptides, the C terminus was more hydrolyzed than the N-terminal ( Fig. 1 ). A large number of peptides were also found generated in this region from Lactobacillus helveticus ( Sadat-Mekmene , 2011a ), Lactobacillus delbrueckii subsp. lactis CRL 581 ( Hebert , 2008 ), Lactobacillus bulgaricus ( Tsakalidou , 1999 ), Lactobacillus lactis subsp. cremoris ( Reid , 1991 ), and S. thermophilus ( Miclo , 2012 ). On the other hand, the N terminus of β-CN was resistant to hydrolysis by B. longum , as previously reported by Chang (2013) . The C terminus of β-CN, which was determined to be a hydrophobic region in this study, is more accessible for hydrolysis by B. longum ( Chang , 2013 ) and by the proteases of S. thermophilus ( Chang , 2014 ; Miclo , 2012 ) and L. helveticus ( Sadat-Mekmene , 2011a ).
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Cleavage sites of peptide bonds on bovine β-, αs1-, and κ-casein hydrolyzed after fermentation with Bifidobacterium longum KACC9156. The dot arrows cleaved peptide bonds in this study. The line arrow cleaved peptide bonds reported by Chang et al. (2013).
For α s1 -CN, the hydrolysis pattern was also consistent with the results of Chang (2013) , who reported that B. longum hydrolyzed the N-terminal region to a greater extent than the C-terminal region. In spite of the similar hydrolysis patterns, the observed cleavage pattern ( Fig. 1 ) was slightly different to the result obtained by Chang (2013) for B. longum , in which we observed relatively more cleavage sites on α s1 -CN. The reason for the different cleavage patterns might be that the structure of α s1 -CN was changed during fermentation in milk to make it more accessible for hydrolysis by B. longum . A similar result was reported by Chang (2014) and Sadat-Mekmene (2011b) , who also found that the proteases PrtS and PrtH of S. thermophilus and L. helveticus , respectively, were more accessible at the N terminus than the C terminus.
In this study, regions found to be resistant to hydrolysis were also identified in the β-CN and α s1 -CN sequences. This may be due to the presence of phosphoserine residues in these regions, which leads to high resistance to hydrolysis ( Chang , 2014 ; Kaspari , 1996 ). In present work, five and eight phosphoserine residues were identified in the regions that were not hydrolyzed on β-CN and α s1 -CN, respectively.
In the case of κ-CN, the cleavage site ( Fig. 1 ) only showed the generation of one peptide, whereas Chang (2013) found two peptides in this region. Furthermore, Zahraa (2010) reported that the glycomacropeptide (f106-169) region, composed of glycan chains, was difficult to hydrolyze because of the presence of hydrophilic amino acids and a negative charge, leading to increased electrostatic repulsion. However, in the present study, only one peptide, f150-151, of this casein was obtained. Fermentation with B. longum could induce a structural change due to weak bond of glycan chains on this glycomacropeptide region that would allow B. longum to access κ-CN for hydrolysis.
As the previously study by Chang (2013) who reported that no peptide detected from α s2 -CN from 0.1% CN solution in 0.05 M sodium phosphate buffer (pH 7.0), none of the peptides identified were found in <3 kDa fraction from fermentate. This observation could be explained by the results of Tauzin (2002) , who found a protected region due to the formation of a tetrameric complex from CN that was more resistant to hydrolysis. Miclo (2012) also suggested that accessibility of this region depends on its protein structure change.
Milk proteins play role precursors to release many peptides relating biological activity ( Korhonen, 2009 ), i.e., ACE inhibitory peptide as this work.
Thirty two peptides relating ACE inhibitory obtained from casein have been reported in literature ( Table 3 ). Out of 28 peptides generated from bovine casein during fermentation in present study, only 3 peptides from β-CN identified through MS/MS analysis (ESI-Q-TOF), YQEP-VLGPVRGPFPIIV, QEPVLGPVRGPFPIIV, GPVRGPFPIIV corresponded to ACE inhibitory bioactive peptides ( Table 3A ). These peptides were consistent with the previously studies reviewed by Yamamoto (1994) , Gobbetti (2002) and Gomez-Ruiz (2002) , respectively. However no ACE inhibitory peptide released from other casein reported in previously study was present. These 3 peptides identified in this study were known the antihypertensive peptide in the literature. Thus, ACE inhibitory activity shown in Table 1 might result from these peptides.
Antihypertensive and potential antihypertensive peptides generated by fermentation withBifidobacterium longumKACC91563 after 24 h at 37℃ (Continued)
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aPeptides obtained from the fermentates in milk with B. longum in the present study.
Actually, peptides which have the high ACE inhibitory activity contain several amino acids (Trp, Phe, Tyr, or Pro) at the extremity of C-terminal and Ala, Val, IIe and Ser called aliphatic amino acids at the N-terminal ( Jao , 2012 ). The three peptides, YQEPVLGPVRGPFPIIV, QEPVLGPVRGPFPIIV, GPVRGPFPIIV identified in present study contain these amino acids except for 4 amino acids, Gln, Glu, Leu and Gly. Thus, from these results, the peptides identified were reasonable to show the antihypertensive activity.
Other peptides as potential antihypertensive peptides which were included the fragment having ACE inhibitory activity were also identified in this study. These peptides were listed in Table 3B displaying 6 for β-CN and 9 for α s1 -CN. Peptide, RELEELNVPGEIVE (f1-14) released from β-CN identified in present study contains LNVPGEIVE reviewed by Gobbetti (2000) as ACE inhibitory peptide. Another peptide, YQEPVLGPVRGPFP (f193-206) from β-CN, contains also the ACE inhibitory peptide, YQEPVLGPVR ( Rokka , 1997 ). The others from β-CN, the fragment GPVRGPFPIIV reviewed by Gomez-Ruiz (2002) as antihypertensive peptide was included in 4 peptides, EPVLGPVRGPFPIIV, PVLGPVRGPFPIIV, VLGPVRGPFPIIV, LGPVRGPFPIIV.
For peptides obtained from α s1 -CN, the 9 peptide, APSFSDIPNPIGSENSEKTTMPLW (f176-199), SFSDIPNPIGSENSEKTTMPLW (f178-199), FSDIPNPIGSENSEKTTMPLW (f179-199), SDIPNPIGSENSEKTTMPLW (f180-199), DIPNPIGSENSEKTTMPLW (f181-199), IPNPIGSENSEKTTMPLW (f182-199), PIGSENS EKTTMPLW (f185-199), IGSENSEKTTMPLW (f186-199), SENSEKTTMPLW (f188-199) was released ( Table 3B ). These peptides contain the peptide TTMPLW (f154-199) which have reviewed that this peptide has a biological activity to inhibit ACE ( Maruyama and Suzuki 1982 ; Pihlanto-Leppälä , 1998 ). The antihypertensive peptide, SDIPNPIGSENSEKTTMPLW (f180-199) occurring naturally in milk, was also reviewed by Islam (2014) .
From our results, these 15 peptides obtained from β-CN and α s1 -CN during fermentation with B. longum may be presented the antihypertensive activity ( Table 3B ) during digestion in vivo . Chang (2014) have reported that some peptides containing bioactive peptide fragment could be released during digestion. To verify whether these peptides show the ACE inhibitory activity as novel antihypertensive peptide after synthesis of these peptides, whether these peptides during digestion with gastro-intestinal enzyme i.e., pepsin, trypsin, chymotrypsin, pancreatin etc. will be short and generated as antihypertensive peptide reviewed in literature and also whether peptides generated during digestion will be novel antihypertensive peptide, the further study will need.
Antihypertensive activity was demonstrated in the low-molecular-weight fraction (<3 kDa) obtained from the fermentate after milk fermentation with B. longum . This fraction was used to identify the CN-derived ACE inhibitory peptides. Using mass spectrometry analysis, three peptides showing antihypertensive activity and 15 peptides with potential antihypertensive activity were identified from CN. Thus, our results suggest that, given its capacity to generate antihypertensive peptides, B. longum KACC 91563 could be used as a starter culture with other lactic acid bacteria in the dairy industry and/or these peptides could be used in functional food manufacturing as antihypertensive agents, owing to their beneficial effects for human health.
This study was sponsored by Postdoctoral Fellowship Program and Research Grant (PJ008585) from National Institute of Animal Science (NIAS), Rural Development Administration of Korea (RDA) of Republic of Korea.
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