Scholarly article on topic 'Milk-derived angiotensin-I-converting enzymeinhibitory peptides generated by Lactobacillus delbrueckii subsp. lactis CRL 581'

Milk-derived angiotensin-I-converting enzymeinhibitory peptides generated by Lactobacillus delbrueckii subsp. lactis CRL 581 Academic research paper on "Animal and dairy science"

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Academic research paper on topic "Milk-derived angiotensin-I-converting enzymeinhibitory peptides generated by Lactobacillus delbrueckii subsp. lactis CRL 581"

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Josefina M. Villegas, Gianluca Picariello, Gianfranco Mamone, Maria Beatriz Espeche Turbay, Graciela Savoy de Giori and Elvira Maria Hebert*

Milk-derived angiotensin-I-converting enzyme-inhibitory peptides generated by Lactobacillus delbrueckii subsp. lactis CRL 581

Abstract: Several strains of Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis were evaluated for their ability to release angiotensin-I-converting enzyme (ACE) inhibitory peptides from a-casein (a-CN) and p-casein (p-CN). Casein peptides resulting from L. delbrueckii subsp. lactis CRL 581-mediated hydrolysis exhibited the highest ACE-inhibitory (ACEI) activities, with values of 53 and 40% for a-CN and p-CN, respectively. The casein hydrolysates were fractionated by reversed-phase high pressure liquid chromatography and some of the active peptides were identified by mass spectrometry. The fraction with the highest ACEI activity arose from p-CN and contained a mixture of the p-CN f194-206 (QEPVLGPVRGPFP) and f198-206 (LGPVRGPFP) peptides. Furthermore, the ACEI tripeptide IPP was identified in all p-CN hydrolysates; L. delbrueckii subsp. lactis CRL 581 produced the highest amount of this peptide. The bioactive peptides released by CRL 581 strain may be used in the formulation of functional foods and nutraceuticals, representing a healthier and natural alternative for regulating blood pressure.

Keywords: lactic acid bacteria, proteinase, ACE-inhibitory peptides, casein

*Corresponding author Elvira María Hebert: CERELA-CONICET. Chacabuco 145, 4000 San Miguel de Tucumán, Tucumán, Argentina. Tel/FAX: +54 381 4310465/4005600. Josefina M. Villegas, Maria Beatriz Espeche Turbay, Graciela Savoy de Giori: Centro de Referencia para Lactobacilos (CERELA) CONICET-Chacabuco 145 - 4000 S. M. de Tucumán -Argentina Gianluca Picariello, Gianfranco Mamone: Istituto di Scienze dell'Alimentazione CNR, Via Roma 64, I-83100 Avellino, Italy

1 Introduction

Milk is among the most widely consumed food by humans. It contains components that provide key nutritive elements, immunological protection, and biologically active substances. Casein is the main protein component of bovine milk, constituting about 80% (2.7 g/100 g milk) of the total protein fraction. Although individual casein subfamilies have not any established physiological role, peptides released from caseins have been shown to possess several biological functions such as antimicrobial, immunomodulatory, enhancement of mineral absorption, antithrombotic, antihypertensive, opioid and antioxidant activities [1-4]. Food-derived biologically active or functional peptides are peptide sequences arising from food proteins that exert a range of a physiological, hormone-like effect in the body, going well beyond their nutritional value [1]. These peptides usually consist of 3-20 amino acids and can be liberated from the mother protein by proteolysis with specific enzymes or by gastrointestinal digestion [2,3].

The most common application of lactic acid bacteria (LAB) is their use as starter cultures in the manufacturing processes of a wide series of fermented dairy products. Microbial fermentation is one of the major processes to generate bioactive peptides, mainly in the dairy industry, considering that the lactobacilli cell-envelope associated proteinases (CEPs) extensively hydrolyze milk proteins during fermentation [1,4-6]. As milk contains only small amounts of amino acids and short peptides, growth of LAB depends on a complex proteolytic system deputed to obtain essential amino acids from caseins during their growth in milk. This complex proteolytic machinery consists of a CEP, which is involved in the first step of casein degradation releasing mainly peptides, as well as of peptide transport systems and several different cytoplasmic peptidases that further cleave the incoming peptides into shorter peptides and amino acids, thus supplying degraded organic nitrogen to the cell [1,7].

"a'"™'1 © 2014 Josefina M. Villegas, et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

Expectedly, the choice of the proteolytic enzymes or strains used for food processing has a crucial impact on the composition of the released peptides [8]. Thus, the strain selection to produce bioactive peptides from fermented milk will depend on the proteolytic activity of the starter culture. Because of its properties, the proteolytic system of lactococci has been the subject of intensive biochemical and genetic research. However, limited information is available on the proteolytic activity of lactobacilli [7]. The use of LAB proteolytic enzymes, traditionally used for the production of different fermented milk products and cheeses, is an interesting alternative for the production of new functional foods containing bioactive peptides such as antihypertensive peptides.

Angiotensin-I-converting enzyme (ACE; peptidyl-dipeptide hydrolase; EC is a multifunctional ectoenzyme located in different tissues. ACE is a nonspecific dipeptidyl carboxypeptidase that critically affects blood pressure, regulating the renin-angiotensin system. This enzyme converts angiotensin I to angiotensin II, increasing blood pressure and aldosterone, while inactivating the vasodilator bradykinin [9]. Therefore, ACE inhibition results mainly in an antihypertensive effect but may also influence different regulatory systems involved in modulating blood pressure, immune defense, and nervous system activity [10]. Peptides with ACE-inhibitory (ACEI) activity have been matter of many studies and industrial applications; several antihypertensive peptides resulting from the casein hydrolysis by different LAB strains have been reported [1,4,11-13].

The proteolytic activity of Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis has been previously characterized [14,15]. These thermophilic microorganisms possess a high proteolytic efficiency compared to other LAB. In this work, we characterized the caseinolytic specificity of four strains of thermophilic lactobacilli as well as their capabilities to release bioactive peptides with ACEI activity from a-casein (a-CN) and p-casein (p-CN). In addition, some of these peptides were isolated, sequenced by tandem mass spectrometry and their bioactivity was characterized.

2 Materials and Methods

2.1 Microorganisms, media, and growth conditions

The following strains of thermophilic lactobacilli were used in this study: L. delbrueckii subsp. lactis CRL 581, L. helveticus CRL 1062, CRL 1177 and CRL 1179. All strains were

obtained from the Centro de Referencia para Lactobacilos (CERELA, Tucuman, Argentina).

Working cultures of lactobacilli were routinely grown in the peptide-rich medium MRS broth (Biokar Diagnostics, France) at 40°C for 16 h. To eliminate carryover nutrients, the cells were harvested by centrifugation at 8,000 x g for 15 min, washed twice with sterile 50 mM sodium phosphate buffer (pH 7.0), and resuspended in the original volume. This cell suspension was used to inoculate the chemically defined medium (CDM) [16], at an initial optical density at 560 nm (OD560) of 0.07. Cells were incubated at 40°C, harvested by centrifugation at OD560 = 0.70, washed twice with saline solution containing 5 mM CaCl2 and resuspended to a final OD560 of 10 in 50 mM sodium phosphate buffer (pH 7.0).

2.2 Proteinase activity assay

Proteinase activity was measured in 50 mM phosphate buffer (pH 7.0) at 40°C, with the chromogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-p -nitroanilide (S-Ala; Sigma), as described by Espeche Turbay et al. [17]. One unit of proteinase (UE) was defined as the amount required to liberate 1 nmol of nitroaniline per min. Specific activity was expressed as UE per mg protein. The protein concentration was determined using the Bio-Rad protein assay according to the instructions of the manufacturer (Bio-Rad Laboratories, Richmond, CA). The Bio-Rad protein assay is based on the Bradford dye-binding method [18].

2.3 Casein hydrolysis

Casein degradation was carried out following the protocol described by Hebert et al. [16]. Washed whole cells (OD560= 10) were incubated with 3 mg ml-1 of substrate a- and ß-CN (Sigma), previously dissolved in 100 mM phosphate buffer (pH 7.0), at a 1:1 (vol/vol) ratio at 40°C for 4 h. Cells were removed by centrifugation, and the supernatants containing different peptide fractions were stored at -20°C for further analysis (RP-HPLC separation, MS analysis of peptide mixtures and ACEI activity). Casein hydrolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [19], RP-HPLC and mass spectrometry (MS) analysis. Protein bands on the SDS-PAGE gels were visualized by silver staining (Bio-Rad). To assess the enzymatic hydrolysis of the substrate as1-CN (f1-23), washed whole cells (OD560 = 10) were mixed with 8 mg ml-1 of the substrate dissolved in 100 mM sodium phosphate (pH 7.0) at a ratio of 1:1 during 4 h at 40°C. Then, the samples were centrifuged (10,000 x g

for 10 min at 4°C) and the supernatants were analyzed by RP-HPLC and MS.

2.4 RP-HPLC separation and MS analysis of peptide mixtures

HPLC analyses were carried out using an Agilent 1100 series HPLC modular system equipped with a Jupiter Proteo 4 (Phenomenex, Torrance, CA, USA) C reversed-phase column (2.1 mm d.i. x 250 mm, 4 ^m particle size). Peptides were separated at a constant flow-rate of 0.2 ml min-1 applying a linear gradient from 5 to 70% solvent B after 5 min of isocratic elution at 5% B. Solvent B was acetonitrile/0.1% (vol/vol) trifluoroacetic acid (TFA) and solvent A was 0.1% TFA in HPLC-grade water. Column effluents were monitored by UV detection at 220 and 280 nm. Approximately 50 ^g of peptide mixture was injected for each run. Fractions of three RP-HPLC runs were manually collected, pooled, freeze dried, and re-dissolved in 200 ^l of water. These peptide fractions were analyzed by MS and their effect on ACE activity was studied.

The peptides were analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight (MALDI-TOF) MS on a Voyager DE-Pro spectrometer (PerSeptive BioSystems, Framingham, MA, USA) equipped with a N2 laser (A = 337 nm), using a-cyano-4- hydroxycinnamic acid as the matrix (10 mg ml-1 in 50% acetonitrile/0.1% TFA). Mass spectra were acquired in the positive reflector or linear ion mode using Delayed Extraction (DE) technology, typically accumulating 200 laser pulses. The accelerating voltage was 20 kV. Mass spectra were externally calibrated through a separate analysis of a standard peptide mixture (PerSeptive Biosystems).

Tandem MS (MS-MS) data were obtained by using a Q-STAR MS (Applied Biosystems, Foster City, CA) equipped with a nanospray source (Protana, Odense, Denmark). Samples were desalted by using ZipTip C18 microcolumns (Millipore, Billerica, MA, USA), and sprayed from gold-coated "medium-length" borosilicate capillaries (Protana). The capillary voltage used was 800 V. Preferentially, doubly-charged ions were selected using the quadrupole mass filter and then induced to fragment by collision at a 20-30 arbitrary units of collision energy. In the case of very small and/ or base-less peptide signals, when the doubly-charged ions were not detected or below a threshold of 15 counts/spectrum, singly-charged ions were selected as the precursors [20]. The collision-induced dissociation spectra were processed with the Analyst 1.1 software (Applied Biosystems).

2.5 ACE activity and inhibition

ACE activity was measured following the method described by Vermeirssen et al. [9]. Furanacryloyl-phenylalanyl-glycyl-glycine (FAPGG) and rabbit-lung extract were used as substrate and enzyme source, respectively. The rabbit-lung ACE was prepared by dissolving 1 g of rabbit lung acetone powder in 10 ml of 50 mM potassium phosphate buffer, pH 8.3; then, the enzyme extract was ultracentrifuged at 40,000 x g for 40 min. Prior to assay, the supernatant was diluted 10-fold with 50 mM potassium phosphate buffer, pH 8.3 (ca. 3 U mg-1 of protein) [21]. Ninety microliters of FAPGG (1 mM), dissolved in 50 mM Tris-HCl buffer (pH 8.3) containing 400 mM NaCl, and 55 ^l of a peptide fraction, or alternatively a synthetic peptide, a reference ACE-inhibitor (e.g. captopril) or water were mixed and pre-incubated for 2 min at 37°C in a microplate reader Cary® 50 UV-Vis Spectrophotometer (Varian, Australia). After addition of 55 ^l of the diluted rabbit-lung acetone extract, the reaction mixture was incubated at 37°C and the decrease in absorbance at 340 nm was measured over a time interval of 30 min. Percent (%) inhibition was calculated based on a standard curve prepared from several dilutions of the enzyme preparation [9]. The ACE-inhibitor captopril (Sigma) was used as a reference ACEI substance at a range of concentrations from 1 pM to 100 nM. Percent inhibition was calculated as follows: (B - A) / (B - C) x 100; where A is the slope of the curve at 340 nm in the presence of both ACE and the peptide fraction, B is the slope of the curve at 340 nm without the peptide fraction, and C is the slope of the curve at 340 nm without ACE.

2.6 Statistical analysis

Results are mean values and standard deviations of three repetitions of three independent assays. Statistical analysis was performed using Minitab 14 software (PA, USA). Comparisons were accomplished by ANOVA general linear model followed by Tukey's pat-hoc test and P < 0.05 was considered significant.

3 Results and Discussion

3.1 Hydrolysis of a- and ß-casein

In a previous work, the CEPs of several thermophilic lactobacilli strains were investigated for their action on the chromogenic substrate S-Ala, as well as by SDS-PAGE using a-CN and ß-CN as substrates [14]. These lactobacilli strains

were classified into four groups, according to the degree of similarity in the degradation pattern of a-CN and p-CN [14]. In this study, one representative strain of each group was selected in order to analyze its caseinolytic specificity as well as its ability to generate antihypertensive peptides from a-CN and p-CN hydrolysis. The selected strains were L. delbrueckii subsp. lactis CRL 581, L. helveticus CRL 1062, CRL 1177 and CRL 1179, with the CRL 581 strain exhibiting the highest proteinase activity.

The proteinases of Lactococcus lactis have been divided into two main groups according to their substrate specificity [7,22]. PI-type proteases hydrolyze predominantly p-CN and, to lesser extent, K-casein (k-CN), while PIII-type proteases degrade a-CN, p-CN and k-CN to a comparable extent [7,22]. The selected lactobacilli strains were able to hydrolyze, in different extent, both a-CN and p-CN [6]. Contrariwise, k-CN was hardly degraded by the studied lactobacilli (data not shown), suggesting that the corresponding CEPs displayed intermediate traits between PI- and PIII-type.

On the other hand, the lactococcal CEPs are classified in seven groups (from a to g) according to their specificities toward the as1-CN fragment comprising residues 1 to 23 [23]. In order to classify the CEPs of the selected lactobacilli, their ability to cleave internal sites of the 1-23 region of the as1-CN was analyzed (Figures 1 and 2). All strains showed different as1-CN (f1-23) degradation patterns, suggesting that the caseinolytic specificity is strain dependent. By characterizing the peptide sequence in these hydrolysates, a pattern of as1-CN (f1-23) breakdown was defined for L. delbrueckii sus1bsp. lactis CRL 581, L. helveticus CRL 1062, CRL 1177 and CRL 1179 (Figure 2). Peptides RtP2, Q9G10L11P12,

Q9G10L11P12Q13, G10L11P12Q13 ^ V15L16N17 ^^ c°mm°n to

all the hydrolysates. In addition to the already identified cleavage sites for L. helveticus CRNZ32, CNRZ 303, CP790, L89, Streptococcus thermophilus and Lc. lactis in the asl-CN (f1-23), such as ^ K^, Q9QM, Q13E14, LtóN17, N17E18 and L21R22, the CEPs investigated in this study were able to hydrolyze the P2I3, L1-P12, P12Q13, E14V15, E18N19 and N19L20 sites, which have never been identified for any of the previously described CEP [24-27]. Therefore, the specificity of the studied lactobacilli proteinases toward the as1-CN (f1-23) fragment did not fit into the criteria for none of the groups cataloguing the lactococcal CEP variants characterized so far [25]. Thus, the analyzed CEPs could be classified as a mixed-type CEPI/ffl variant. On the other hand, these results confirm the different caseinolytic specificity of each CEP analyzed in this study.

5e+5 4e+5 3e+5 2e+5 1 e+5 -C

-1e+5 5e+5 --

4e+5 -

3e+5 -

2e+5 -

1e+5 C

-1e+5 5e+5

4e+5 -

3e+5 -

2e+5 -

1 e+5 -

-1e+5 -5e+5 -

4e+5 -

3e+5 -

2e+5 -

0 10 20 30 40 50 60

Time (min)

Figure 1: Analytical reverse-phase HPLC patterns of the products of as1-CN (f1-23) degradation by the CEP of L. delbrueckii subsp. lactis CRL 581 (B), L. helveticus CRL 1062 (C), CRL 1177 (D) and CRL 1179 (E). Non-inoculated as1-CN (f1-23) was used as control (A).

3.2 Characterization and isolation of ACE-inhibitory peptides

Proteinases of LAB can hydrolyze more than 40% of the a-CN and p-CN peptide bonds, producing more than 100 different peptides [22,28]. Therefore, LAB could potentially generate various casein-derived bioactive peptides with

antihypertensive, antimicrobial, or immunomodulating effects [29-31]. Several studies reported the synthesis of ACEI peptides in pasteurized Calpis™ sour milk fermented by both L. helveticus and Saccharomyces cerevisiae [32-34].

A: CRL 581

5 10 15 20


B: CRL 1062

5 10 15 20

rpkhpikhqglpqevlnenllrf j— « ч ::=_» «

C: CRL 1177

rpkh^ikhqSlpqe^ lnenTlrf

D: CRL 1179


Figure 2: Locations of the peptides (double-ended arrows) identified in the primary sequences of as1-CN (f1-23) and released by CEP from L. delbrueckii subsp. lactis CRL 581 (A), L. helveticus CRL 1062 (B), CRL 1177 (C) and CRL 1179 (D).

L. helveticus and L. delbrueckii subsp. lactis, both important industrial strains, are used as starter cultures for the manufacture of fermented dairy products and several types of cheeses. Because several authors demonstrated that many ACEI peptides derive from caseins [2,35], we selected the L. delbrueckii subsp. lactis CRL 581, L. helveticus CRL 1062, CRL 1177 and CRL 1179 CEPs to evaluate their ability to produce antihypertensive peptides. The ACEI activity percentages of the four a-CN and p-CN hydrolysates are shown in Table 1. The highest levels of ACE inhibition corresponded to L. delbrueckii subsp. lactis CRL 581, with values of 53 and 40% for a-CN and p-CN, respectively. Considering these results, the hydrolysate of L. delbrueckii subsp. lactis CRL 581 was selected for further analysis. The hydrolysis of a-CN and p-CN by the CEP of L. delbrueckii subsp. lactis CRL 581 after 4 h of incubation at 40°C was analyzed by RP-HPLC (Figure 3). These protein fractions were highly degraded by the CRL 581 strain, showing different peptide profiles. Thirty-three and 32 peptide fractions in a-CN and p-CN hydrolysates, respectively, were isolated by RP-HPLC (Figure 3), identified by MS analysis and individually assayed for their ACEI activity. The ACEI index of the most active fractions, together with their retention time, is summarized in Table 2. In particular, fraction 1 and 3, derived from a-CN and fraction 20 and 21, from p-CN, showed the highest ACEI potential, with values above 90%. The low antihypertensive activity observed in some fractions could be due to a poor recovery aside from a low enzymatic inhibition (Figure 3 and Table 2).

3.3 Identification of peptides

Peptides contained in the ACEI active fractions were identified by MS and tandem MS and are assigned in Table 2. Their sizes varied from 3 to 13 amino acids. Fraction 1 of a-CN contained a mixture of very short peptides, thus its identification was not possible. On the other hand,

Table 1: ACE-inhibitory activities of a-CN and p-CN hydrolysates generated by the CEP of thermophilic lactobacilli

Strain ACE inhibition (%)a

a-CN hydrolysate ß-CN hydrolysate

L. delbrueckii subsp. lactis CRL 581 53 ± 5 40 ± 5

L. helveticus CRL 1062 12 ± 2 3 ± 1

L. helveticus CRL 1177 9 ± 1 2 ± 0.5

L. helveticus CRL 1179 8 ± 2 5 ± 1

"Values are the means ± standard deviations of the results from three independent experiments. Captopril 8 nM was used as control (100% of inhibition).

Figure 3: RP-HPLC profiles of the peptides from the 1%-TFA-soluble fraction obtained after 4 h of hydrolysis of a-CN (A) and p-CN (B) by the action of CEP from L. delbrueckii subsp. lactis CRL 581.

fraction 20, derived from p-CN, consisted of p-CN f194-206 and f198-206 peptides. Interestingly, these peptides arise from one of the p-CN regions that are considered "strategic" under the standpoint of the biological activity. Recently, these C-terminal peptides of p-CN have been proposed as possible candidates to exert bioactivity in vivo, considering that they appear to be released in vivo in piglets and to survive the gastrointestinal digestion [36]. ACEI assays with peptides having varying C- or N-terminal amino acids, have demonstrated that the nature of the C-terminal amino acid is crucial to the overall binding of the peptide to the active site of the enzyme. This enzyme generally prefers substrates or inhibitors that contain mainly aromatic (W, Y, F), aliphatic (I, A, L, M) aminoacids or proline at the C-terminal position, or aromatic (Y, F), aliphatic (V, I, A) amino acids or arginine

in the penultimate position [37,38]. This would explain the ACEI effect recorded for p-CN (194-206) QEPVLGPVRGPFP and (198-206) LGPVRGPFP peptides, in which the three C-terminal amino acids are hydrophobic. In addition, the tripeptide IPP was identified in the fraction 21 from the p-CN hydrolysate, which is known as one of the strongest inhibitors of ACE within peptides derived from milk proteins [1-3,33]. Several clinical trials showed that consecutive uptakes of IPP for more than a week resulted in significant reduction of blood pressure in humans [39-42]. In order to establish if the p-CN hydrolysates derived from the other studied strains contained the peptide IPP, we monitored the presence of positive ions with a molecular mass of 326.30 Da by MS. The tripeptide was detected in all samples, though with variable quantitative levels among strains (data not shown).

Table 2: Peptides identified in the a-CN and p-CN hydrolysates produced by the CEP of L. delbrueckiisubsp. lactis CRL 581

Fraction8 Retention time (min) ACE inhibition (%) Peptide identityb Sequence Mass (Da) Measured Expected

alpha 1 2.2 90.55 nd nd

alpha 2 3.3 13.99 nd nd

alpha 3 4.6 91.85 nd nd

alpha 4 5.7 11.83 nd nd

alpha 11 22.0 13.99 as1-CN (83-89) KEDVPSE 803.38 802.37

alpha 12 23.0 16.14 as1-CN (125-128) QGIH 454.19 453.22

alpha 13 23.5 13.99 as1-CN (10-14) GLPQE 543.27 542.27

alpha 18 28.0 9.68 as1-CN (76-84) VEQKHIQKE 1137.61 1138.29

alpha 19 30.8 9.68 as1-CN (110-121) EIVPNSAEERLH 1473.64 1472.67

beta 1 6.1 13.88 nd nd

beta 11 30.0 13.88 p-CN (157-161) FPPQS 575.29 574.28

beta 20 42.2 98.61 p-CN (194-206) QEPVLGPVRGPFP, 1392.73 1391.76

p-CN (198-206) LGPVRGPFP 939.51 938.53

beta 21 46.2 95.01 P-CN (74-76) IPP 325.2 326.2

aNumbers refer to collected RP-HPLC fractions. The not mentioned fractions had no activity against ACE. Intact casein (time 0 h) had a retention time of approximately 55 min.

bNumbers refer to amino acid positions in the as1-CN and p-CN mature protein sequence.

As expected, the p-CN hydrolysate generated by L. delbrueckii subsp. lactis CRL 581 showed the most intense signal, being 3, 4 and 36 times higher than those obtained from L. helveticus CRL 1062, CRL 1177 and CRL 1179 hydrolysates, respectively.

The data presented in this study contribute to enlarge the limited knowledge on thermophilic lactobacilli CEPs. To our knowledge, this is the first work that uses a L. delbrueckii subsp. lactis strain to produce antihypertensive peptides from caseins. Therefore, L. delbrueckii subsp. lactis CRL 581 could be used as a starter culture for the development of a novel functional food designed for controlling the blood pressure of persons suffering from moderate hypertension.

Acknowledgements: The authors acknowledge the financial support of CONICET, ANPCyT, MINCyT and FONARSEC from Argentina.

Received: July 26, 2013; Accepted: December 06, 2013


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