Open access peer-reviewed chapter

Antihypertensive Peptides Specific to Lactobacillus helveticus Fermented Milk

By Taketo Wakai and Naoyuki Yamamoto

Submitted: March 2nd 2011Reviewed: November 13th 2011Published: March 14th 2012

DOI: 10.5772/28695

Downloaded: 2060

1.Introduction

Peptides are well known as nitrogen sources to supply various amino acids for many different organisms, and also have many hormonal functions in our body. Previous studies have reported a secondary role for peptides with specific amino acid sequences that possess biological function in vivo(1-8). To prepare biologically active peptides (bioactive peptides), food ingredients containing protein are generally hydrolyzed by some proteolytic enzymes. In particular, milk proteins such as bovine casein and whey proteins have been used for preparations of bioactive peptides because inexpensive and safe sources are readily available.Various physiologically functional peptides, such as immunostimulating peptides (1), antimicrobial peptides (2), opioid peptides (3), mineral soluble peptides (4) andantihypertensive peptides (5-8) and have been isolated from enzymatic hydrolyzates of raw food materials and fermented food products.

Among these bioactive peptides, antihypertensive peptides have been extensively studied and reviewed (5-8). Hypertension is a major risk factor in cardiovascular disease, such as heart disease and stroke. In order to reduce the incidence of disease, pharmacological substances can be used to decrease high blood pressure to within the normal range. Angiotensin I-converting enzyme (kininase II; EC 3.4.15.1) (ACE) is predominantly expressed as a membrane-bound ectoenzyme in vascular endothelial cells and several other cell types, including absorptive epithelia, neuroepithelia and male germinal cells (9, 10). A dipeptidyl carboxypeptidase, ACE catalyzes the production of a vasoconstrictor, angiotensin II, and inactivates a vasodilator, bradykinin (11, 12). The first competitive inhibitors to ACE were reported as naturally occurring peptides isolated from snake venom (13, 14). Then, inhibitory activities on ACE, which plays an important role in blood pressure regulation are generally assessed for preparation of antihypertensive peptides.

Among lactic acid bacteria, Lactobacillus helveticus had the highest extracellular proteinase activity and the highest ability to release the specific antihypertensive peptides in the fermented milk (15). So, this paper mainly reviews processing of the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), by proteolytic enzymes of L. helveticus. The antihypertensive effects of these in vitro and in vivo studies, clinical study, and the mode of action are also reviewed. The antihypertensive effect of the L. helveticus fermented milk compared to that produced by various lactic acid bacteria and proteolytic systems of L. helveticus is reviewed for the discussion in more detail. Finally, processing of VPP and IPP specific to the L. helveticus is discussed in the context of comparative genome analyses of corresponding proteolytic enzymes in various lactic acid bacteria.

1.1. Antihypertensive peptide in fermented milk

1.1.1. ACE inhibitory peptides from milk proteins

Many kinds of ACE inhibitory (ACEI) peptides have been reported from enzymatic hydrolyzates of milk protein, as well as synthetic peptides and fermented products (16-24) (Table 1). Spontaneously hypertensive rat (SHR) is a useful animal model to evaluate the antihypertensive activity of ACEI peptides because the systolic blood pressure of SHR reaches over 230 mmHg and is powerful tool for detection of the in vivo effect. Some of the orally administered ACEI peptides have demonstrated strong antihypertensive effects in SHR (Table 1).

PeptideSourcePreparation(IC50(µM)Dose (mg/kg)(((SBP(mm Hg)
<Enzymatic hydrolysate"/
FFVAPFPEVFGKαs1-caseinTrypsin77100-13.0
AVPYPQR-caseinTrypsin15100-10.0
TTMPLWβs1-caseinTrypsin16100-13.6
LKPNMAldolaseThermolysin2.460-23
LKPAldolaseChicken muscle0.3260-18
IPA-lactogloblinProteinase K1418-31
VYPFPG-caseinProteinase K2218-22
GKP-microglobulinProteinase K3528-26
FP-casein, albuminProteinase K3158-27
YKVPQLαs1-caseinProteinase221-12.5
<Fermented products"/
RFSake leesBrewingND100-17
VWSake leesBrewing1.4100-10
YWSake leesBrewing10.5100-28
VYSakeBrewing7.1100-31
IYPRYSakeBrewing4.1100-19
VPP-caseinFermentation91.6-20
IPP- and κ-caseinFermentation51-15.1
YP βs1, - and κ-caseinFermentation7201-27.4
ND: Not described
(IC50: Peptide concentration that shows 50% inhibition of ACE activity
(((SBP: systolic blood pressure of spontaneously hypertensive rat

Table 1.

Antihypertensive peptides derived from caseins by proteolytic action.

Lactic acid bacteria have proteolytic systems that can hydrolyze milk protein and have been reported to utilize the peptides released from the milk protein casein (25-27). Among lactic acid bacteria, Lactobacillus helveticus had the highest extracellular proteinase activity and the ability to release the largest amount of peptides in the fermented milk (Table 2). As a result, among various kinds of fermented milk, the antihypertensive effect was specific to the L. helveticus fermented milk (15). In our study, an antihypertensive effect related to ACEI peptides was found in sour milk produced by L. helveticus(19, 28). Two ACE inhibitory peptides were purified from sour milk and identified as VPP and IPP. The ACEI activity of the two peptides was very high, 9 M and 5 M, compared to other reported peptides (Table 1). The amino acid sequences of VPP and IPP were found in the primary structure of bovine -casein (84-86) (74-76) and -casein (108-110), respectively. These peptides were produced during fermentation (16), but were not found in the hydrolyzate of casein after digestion with an extracellular proteinase of L. helveticus(29). Oral administration of L. helveticus fermented milk containing VPP and IPP to SHR, with a single dose of 5 ml/kg body weight, significantly decreased systolic blood pressure between 4 and 8 h after administration (28). The antihypertensive effect of these two chemically synthesized peptides was also observed between 2 and 8 h after administration and the effects were dose-dependent. Furthermore, a dose-dependent antihypertensive effect of these two chemically synthesized peptides was also observed from 0.1 to 10 mg/kg of body weight (28).

StrainPeptide conc.Proteinase
act.
ACEI act.Change in SBP
   (%)(U/ml)(U/ml)(mmHg)
Non-fermented milk0.00-0- 5.0 ± 7.3
(Lactobacilli)
L. helveticusCP7900.1923058- 27.4 ± 13.3 **
L. helveticusCP6110.2536770- 20.0 ± 9.6 **
L. helveticusCP6150.1842051- 23.0 ± 13.4 **
L. helveticus JCM10060.1518226- 15.2 ± 9.3 *
L. helveticusJCM11200.1011234- 6.5± 10.8
L. helveticusJCM10040.2118648- 29.3 ± 13.6 **
L. delbrueckii subsp.bulgaricus CP9730.1910522- 0.8 ± 8.2
L. delbrueckii subsp.bulgaricus JCM10020.1112428- 4.5 ± 4.0
L. casei CP6800.01353- 0.2 ± 6.6
L. casei JCM11340.00289- 7.0 ± 11.2
L. casei JCM11360.092518- 9.6 ± 7.2
L. acidophilus JCM11320.00288- 8.7 ± 7.8
L. delbrueckii subsp. lactisJCM11050.081816- 3.3 ± 3.5
(Streptococci)
S. thermophilus CP10070.02253- 2.4 ± 8.1
(Lactococci)
L. lactis subsp. lactis CP6840.00354- 7.3 ± 10.5
L. lactis subsp. cremoris CP3120.02184- 5.8 ± 13.9
Significant differences from the control, p< 0.01, p< 0.05.
ACEI activity: Peptides that show 50% inhibition of ACE activity was defined as one unit.
SBP: systolic blood pressure of spontaneously hypertensive rat

Table 2.

Antihypertensive effects in spontaneously hypertensive rats and ACE inhibitory activities of various fermented milk.

1.2. Clinical effects of the fermented milk 

Hypertension is a major risk factor in cardiovascular diseases, such as heart disease and stroke. In order to reduce the incidence of disease, pharmacological substances can be used to decrease high blood pressure to within the normal range. In the first Japanese study with the fermented milk, hypertensive subjects were randomly assigned to two groups: the one group ingested 95 ml of the milk, containing 3.4 mg of VPP and IPP, daily for 8 wk; the other group ingested the same amount of artificially acidified milk as a placebo, for 8 wk (30). In the fermented milk group, systolic blood pressure decreased significantly between 4 and 8 wk after the beginning of ingestion, but not in the placebo group (30). Moreover, clinical tests were performed for Japanese subjects with different blood pressure levels, which confirmed the mild and prolonged effects for the hypertensive subjects following oral administration of bioactive milk(30-33) (Fig. 1). In a pilot study conducted in Finland, the antihypertensive effect was also observed in the group ingesting L. helveticus fermented milk containing the two tripeptides (34, 35). Moreover, a recent study indicated significant beneficial effects of hypertensive patients ingesting the L. helveticus fermented milk over a long period of time for 21 wk (35). There was a significant decrease in systolic blood pressure (6.7 ± 3.0 mmHg) by comparative study with placebo group.

Figure 1.

Blood pressure lowering effect of Lactobacillus helveticus fermented milk product for subjects with high-normal hypertension (Nakamura et al., J. Nutritional Food (in Japanese) 2004, 7, 123-137). Significant difference from initial value (t-test): p<0.05, p<0.01. Significant difference from placebo group (Bonferroni test): # p<0.05, ## p<0.01.

1.3. Processing of antihypertensive peptides in L. helveticus

1.3.1. Proteolytic system in lactic acid bacteria

Many kinds of proteolytic enzymes have been reported from lactic acid bacteria, and were reviewed extensively(26, 27, 36, 37). The components of the proteolytic systems of lactic acid bacteria are divided into three groups, including the extracellular proteinase that catalyzes casein breakdown to peptides, peptidases that hydrolyze peptides to amino acids and a peptide transport system. The number of proteinases was reported from lactococci, which are mainly used in cheese making. The extracellular proteinase activity is linked to cell growth in milk and seemed to be essential for utilization of milk protein for growth. The gene encoding the proteinases, named prtP, prtH and prtH2, has been sequenced and characterized from several of L. lactis strains (38-40). These proteinases are processed to active enzymes by removal of N-terminal polypeptides from the pre-proteinase with the help of the maturation protein, PrtM.

1.4. Processing by a cell wall-associated proteinase

The first step in casein decomposition is typically caused by an extracellular proteinase, and further digestion to amino acids is catalyzed by many kinds of intracellular peptidases (25, 36). Among the lactic acid bacteria, L. helveticus has the highest extracellular proteinase activity and the ability to release the highest amount of peptides in fermented milk (Table 2). L. helveticus strains were classified into 2 types based on differences in the extracellular proteinase (41). One type has an enzyme with a molecular weight of 170 kDa with homology to the lactococcus enzyme, and other has an enzyme with a molecular weight of 45 kDa (29, 42). Polycloncal antibodies raised against the mature 170 kDa proteinase reacted not only with the 170 kDa enzyme but also the 53 kDa protein (Table 3). The 53 kDa protein was thought to be a degradation product from the 170 kDa active enzyme. A gene encoding a proteinase with a homology to the Lactococcus proteinase was cloned and sequenced from L. helveticus CNRZ32 (43). A gene encoding PrtM gene was also cloned from L. helveticus CNRZ32. On the other hand, the gene encoding a small type of proteinase with a molecular weight of 45 kDa was cloned from L. helveticus CP790 strain (44). A 46 kDa pre-proteinase was activated to the 45 kDa active proteinase by release of 7 amino acids from the N-terminus with the help of a maturation protein (44, 45).

Moreover, a slight difference in the specificity of the two types of proteinases toward casein was suggested for the two types of L. helveticus strains (46). However, there were no clear relationships between proteinase specificity and the antihypertensive effects and the ACEI activities of the fermented milk, which depended on the strain of L. helveticus used. The extracellular proteinase activity of each L. helveticus strain was almost correlated with ACEI activity in the fermented milk (Table 1). These results strongly suggest that the proteolysis of casein by the extracellular proteinase is the most important parameter in the processing of active components. This possibility is also supported by the fact that the L. helveticus CM4, which was selected for its strong proteinase activity compared to other L. helveticus strains, has the ability to release ACE inhibitory peptides in the fermented milk (47). The proposed importance of the proteinase was also supported by the fact that a proteinase negative mutant was not able to generate antihypertensive peptides in the fermented milk, whereas the wild-type strain had the ability to release strong antihypertensive peptides in the fermented milk (16). Strain CM4, which had the highest antihypertensive peptide production in fermented milk, seemed to have the potential for use as a functional food product.

NoStrain SubspeciesReactivity (CP790)1Reactivity (CP53)2Type
1L. helveticus CP39J345 kDa45 kDaA
2L. helveticus CP53H4ND553, 170 kDaB
3L. helveticus CP209J45 kDa45 kDaA
4L. helveticus CP210J45 kDa45 kDaA
5L. helveticus CP293J45 kDa45 kDaA
6L. helveticus CP510J45 kDa45 kDaA
7L. helveticus CP611J45 kDa45 kDaA
8L. helveticus CP615J45 kDa45 kDaA
9L. helveticus CP617J45 kDa45 kDaA
10L. helveticus CP789J45 kDa45 kDaA
11L. helveticus CP790J45 kDa45 kDaA
12L. helveticus JCM1004HND53, 170 kDaB
13L. helveticus JCM1006J45 kDa45 kDaA
14L. helveticus JCM1007JND53, 170 kDaB
15L. helveticus JCM1062JND53, 170 kDaB
16L. helveticus JCM1103HND53, 170 kDaB
17L. helveticus JCM1120HND53, 170 kDaB
(Yamamoto et al., 1998, Biosci.Biotech.Biochem. 58, 776-778)
1Monoclonal antibody to the proteinase from L. helveticus CP790
2Polyclonal antibody to the proteinase from L. helveticus CP53
3Classified as L. helveticusbiovarjugurti
4Classified as L. helveticusbiovarhelveticus
5Not detected

Table 3.

Immunological Difference of Proteinases of L. helveticus Strains with Two Types of Antibodies.

Currently, we completed the whole genome sequence of L. helveticus CM4, and the results revealed the presence of 2,171 open reading frames in 2,028,493 bp of whole DNA sequence (unpublished data). Then, whole genome sequences of CM4 and DPC4571 (48) were compared as shown in Table 4 for full understanding of the intracellular processing to VPP and IPP (49). As shown in Table 4, three genes for cell-wall associated proteinase genes, prtY, prtH2 and prtM2, and 23 kinds of intracellular peptidases were detected in the CM4 sequence. The genes of prtH2 and prtM2 were detected both in CM4 and DPC4571, but the prtM2 was considered to be pseudo-genes for extracellular proteinase in the previous study (50). However, no prtH1 and prtM1 genes reported in CNRZ32 (51) were detected in sequences in both CM4 and DPC4571 strains. The genes of prtH2 and prtM2 were detected both in CM4 and DPC4571, but the prtM2 was considered to be pseudo-genes for extracellular proteinase in the previous study. However, no prtH1 and prtM1 genes reported in CNRZ32 (51) were detected in sequences in both CM4 and DPC4571 strains. On the other hand, one of the cell wall-associated proteinase gene (prtY) corresponding to a 45 kDa proteinase previously detected in L. helveticus CP790 strain (41, 42) was detected in CM4 strain but not in DPC4571 strain (Table 1). These results reveal the cell wall-associated extracellular proteinase which plays key role in decomposition of casein might be different among detected proteolytic enzymes between CM4 and DPC4571. On the other hand, the large size of the proteinase gene (prtH) corresponding to a 200 kDa proteinase and its maturation gene (prtM) reported by Pederson et al.(43)were not detected in the CM4 and DPC4571 genes. This observation supports our previous results that there are two types of extracellular proteinases, and L. helveticus can be classified by the proteinase type as shown in Table 4. This result also suggested that the extracellular proteinase might play a key role to release high amount of VPP and IPP from the comparative analysis of both proteolytic genes (Table 4).

Proteolytic enzymeGeneMolecular weight (kDa)Protein IDIdentity(%)
CM4DPC4571DPC4571
ProteinaseprtY47.0ND--
prtH2181.6180.871-99.2
prtM33.732.7ABX2756398.0
AminopeptidasepepC151.451.4ABX2658298.9
pepC252.950.2ABX2706598.6
pepN95.895.9ABX2773199.4
pepN257.257.2ABX27544100.0
pepA41.340.1ABX2675899.4
XPDAPpepX90.590.6ABX2741999.6
EndopeptidasepepE50.050.0ABX2646699.8
pepE251.450.3ABX2645799.8
pepF68.168.1ABX2768699.3
pepO73.673.5ABX2735899.4
pepO273.873.5ABX2721198.6
pepO373.172.6ABX2643399.7
TripeptidasepepT47.146.7ABX2730599.3
pepT248.848.4ABX2716599.5
DipeptidasepepD154.054.1ABX2762599.4
pepD254.954.9ABX2737599.8
pepD353.553.5ABX27723100.0
pepV51.551.5ABX2722498.9
pepDA53.553.5ABX2649299.6
ProlidasepepQ41.241.2ABX2666499.5
pepQ241.441.1ABX2740599.7
ProlinasepepPN35.035.0ABX2763399.7
ProlineiminopeptidasepepI33.933.8ABX2637599.3
ND: Not detected, *size of the reported pseudo-gene

Table 4.

Genes encoding proteolytic enzymes reported in Lactobacillus helveticusstrains.

By the proteolytic action of the extracellular proteinase in CP790 (and CM4), a long -casein peptide with a 28 amino acid residue including VPP and IPP sequences was generated (29) (Fig. 2). The proteinase activity is easily repressed by accumulated peptides by the proteinase in the fermented milk. Moreover, the enzyme activity is inactivated by pH drop during the fermentation. So, the first degradation of casein by the extracellular proteinase would be occurred mostly at the beginning of the fermentation.

Figure 2.

Postulated proteolytic system for Val-Pro-Pro and Ile-Pro-Pro processing in Lactobacillus helveticus.PrtY: cell-wall proteinase, PrtM: maturation protein, PptO: oligopeptide transporter, PepO: endopeptidase O, PepO2: endopeptidase O2, PepC2: aminopeptidase C2, PepX: X-prolyldipeptidylaminopeptidase. Up- and down-regulation and amount of release are indicated by arrows as () and (), respectively.

1.4.1. Intracellular processing by some peptidases

Next, the long peptide was thoroughly hydrolyzed to shorter peptides by intracellular peptidases. Intracellular peptidases of L. helveticus and these genes were well reviewed, previously (37), however, there was no clear explanation for the processing of VPP and IPP. Long peptide containing two kinds of tri-peptide sequences released by the extracellular proteinase from β-casein will be incorporated into the cell by the oligopeptide transporter (PptO), and processed intracellularly to VPP and IPP by some peptidases (Fig. 2). Carboxyl peptidase most likely needs for the release of C-terminal amino acid from Pro-Pro-X sequence. However, recently, a key enzyme that can catalyzed carboxyl terminal processing to produce VPP and IPP was detected and purified from the CM4 strain (Fig. 2) (52). The enzyme had a homology to an endopeptidase (PepO) from L. helveticus CNRZ32 by amino terminal sequence analysis of the purified enzyme (52) and a homology with the deduced amino acid sequence of the gene (53). The enzyme can catalyze C-terminal processing of VPPFL and IPPLT to VPP and IPP.

Based on the previous reported characteristics of many peptidases from L. helveticus, processing of the N-terminal sequence of VPP and IPP are presumed and summarized in Fig. 2. Generally, amino peptidase shows broad specificity toward amino acid at N-terminal end, however, amino terminal processing seems to terminate if proline residue is present at the N-terminal end in the peptide. However, X-prolyl dipeptidyl aminopeptidase (XPDAP) is able to release the di-peptide with a sequence of X-Pro, from the N-terminus. On the other hand, aminopeptidase may stop the hydrolysis at a Xaa-Pro-Pro- sequence if it is present. Therefore, the N-terminal processing to release VPP and IPP may be catalyzed by specific aminopeptidases, such as pepC2 and XPDAP as shown in Fig. 2. However, for more detailed understanding of these peptide processingin L. helveticus, productivities of these peptides in transformant strains expressing the each postulated peptidase gene or disrupting of these corresponding peptidase genes from wild type strain should be studied.

1.5. Comparison of the L. helveticus proteolytic system to those in other lactic acid bacteria

The unprocessed proteinase of L. lactis consists of about 1950 amino acid residues, and the mature proteinase of L. lactis is a serine type enzyme with a molecular mass between 180-190 kDa (38-40). The gene encoding the proteinase, named prtP, has been cloned and sequenced from several of L. lactis strains (38-40). These proteinases are processed to active enzymes by removal of N-terminal polypeptides from the pre-proteinase with the help of the maturation protein, PrtM. Recent decay, whole genome sequences for more than 36 kinds of lactobacilli have been reported. The comparative analysis of those genes to other species revealed that the CM4 peptidases were more homologous to those in Lactobacillus acidophilus NCFM strain (54)(Table 5). Moreover, homologies of CM4 peptidases were detected with peptidases in Lactobacillus gasseri ATCC33323 (55) and Lactobacillus johnsonii (NC533) (56). These observations suggest that the L. acidophilus group has similar peptidases, endopeptidase, XPDAP, and aminopeptidase to L. helveticus CM4 and might have the ability to process VPP and IPP if the initial decomposition of casein by an extracellular proteinase is accelerated in the fermentation process.

1.6. Repression of proteolyticsystemg

For growth of lactic acid bacteria in milk, the proteolytic system is activated in the milk medium because of alimited amount of amino acids. However, during fermentation in the milk medium,the proteolytic system of lactic acid bacteria is repressed by accumulated peptides in the fermented milk. The amount of VPP and IPP in the L. helveticus fermented milk was also repressed if amino acids were added to the fermented milk (57). Microarray analysis of the whole L. helveticus CM4 genome suggested extracellular proteinase, endopeptidases, XPDAP, some aminopeptidases and some kinds of peptide transporters might be suppressedby addition of amino acids and be involved in the processing of the VPP and IPP (Fig. 2). Regulatory systems (57) that repress the proteolytic enzymes and transporters in the presence of amino acids was reported as codY system in Lactococcus lactis (58, 59). However, there is no codY-like protein in theLactobacillus genome that has been reported. Thus, a novel type regulatory system for the proteolytic system must exist in lactobacilli and strongly affect on the release of VPP and IPP in L. helveticus.

Table 5.

Proteolytic enzymes in various lactic acid bacteria with homology to enzymes in Lactobacillus helveticus CM4, which are expected to have roles in processing Val-Pro-Pro and Ile-Pro-Pro.

2. Conclusion

In this paper, we showed the potential of bioactive peptides to maintain blood pressure in the normal range. About 30% of Japanese people are estimated to be at risk for hypertension. Generally, hypertension has been improved by medication and partly by controlling the diet. Recently, some food products containing antihypertensive peptides and proven antihypertensive effects in clinical studies were recognized as functional foods, Foods for Specified Health Use (FOSHU) in Japan. Biologically functional peptides exerting a mild influence on hypertensive subjects without adverse effect have enormous potential in reducing the risk of cardiovascular disease.

Among many kinds of commercially available lactic acid bacteria, L. helveticus has ability to release functional peptides such as antihypertensive peptides in the fermented milk. These potential is strongly depends on the activities of proteolytic enzymes of L. helveticus. So, the isolation and mutation breeding of a new L. helveticus strain having strong ability to release peptides in the fermented milk is very important to develop industrially useful fermented milk. For the understanding of the antihypertensive peptides, based on comparative analysis with other Lactobacillus proteolytic enzymes, the extracellular proteinase and endopeptidase enzymes seemed to be unique to L. helveticus and seemed at least partially explain the L. helveticus specific release of VPP and IPP from fermented milk. For a full understanding of protein processing, the genomic information and the analysis of the L. helveticus will be a very useful tool and might be needed in future studies.

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Taketo Wakai and Naoyuki Yamamoto (March 14th 2012). Antihypertensive Peptides Specific to Lactobacillus helveticus Fermented Milk, Biotechnology, Reda Helmy Sammour, IntechOpen, DOI: 10.5772/28695. Available from:

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