Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis
Introduction
It is now accepted that enzymatic hydrolysis of food protein releases peptides that may exhibit different biological activities. These protein fragments, known as bioactive peptides, can be released from the precursor inactive protein during gastrointestinal digestion or during food processing (Meisel, 1997). Among the different groups of bioactive peptides, angiotensin converting enzyme-inhibitory (ACEI) peptides are receiving special attention due to their beneficial effects related to hypertension and to the high incidence of cardiovascular diseases related to hypertension in developed countries.
The formation of ACEI peptides by enzymatic hydrolysis (Hernández–Ledesma, Recio, Ramos, & Amigo, 2002; Pihlanto-Leppälä, Koskinen, Piilola, Tupasela, & Korhonen, 2000), by milk fermentation (Gobbetti, Ferranti, Smacchi, Goffredi, & Addeo, 2000; Leclerc, Gauthier, Bachelard, Santure, & Roy, 2002; Nakamura, Yamamoto, Sakai, & Takano, 1995a; Robert, Razaname, Mutter, & Juillerat, 2004) or cheese ripening (Gómez-Ruiz, Ramos, & Recio, 2002; Saito, Nakamura, Kitazawa, Kawai, & Itoh, 2000) has been extensively reported. However, only few studies have demonstrated the effect of specific peptide sequences on hypertensive animals. Nakamura, Yamamoto, Sakai, and Takano (1995a) isolated and identified two ACEI peptides valine–proline–proline and isoleucine–proline–proline (VPP and IPP) derived from casein in milk fermented with a starter containing Lactobacillus helveticus and Saccharomyces cerevisiae. These two peptides have also been found in milk fermented with Lb. helveticus LBK-16 H (Sipola, Finckenberg, Korpela, Vapaatalo, & Nurminen, 2002). These peptides exhibited potent ACEI activity and proven antihypertensive effects in both animals and humans (Hata et al., 1996; Nakamura, Yamamoto, Sakai, & Okubo et al., 1995b; Seppo, Jauhiainen, Poussa, & Korpela, 2003; Sipola et al., 2001).
Difficulties in peptide identification limit the knowledge of bioactive peptide formation and release from the precursor proteins. Several chromatographic steps are often necessary to purify the peptides of interest and these are finally identified by mass spectrometry (MS) alone or combined with N-terminal sequence analysis. The development of routine and reliable liquid chromatography-MS instruments made it possible to analyse peptides included in complex mixtures without needing to separate the individual components (Papayannopoulos, 1995). These analytical techniques can be of invaluable help when identifying bioactive peptides from complex substrates such as food protein hydrolysates or fermented products.
In a previous paper, (Muguerza et al., 2006), we have isolated four Enterococcus faecalis strains from raw milk that were able to produce fermented products with potent ACEI activity and antihypertensive activity in spontaneously hypertensive rats (SHR). These activities could not be attributed to peptides VPP and IPP because these two peptides were absent in these fermented products. The aim of the present study was to identify the peptides responsible for the antihypertensive activity found for the fermented milk produced with these strains of E. faecalis. Isolation of the peptides was performed by following two chromatographic steps and they were sequenced by tandem-MS. The antihypertensive effect of peptides that exhibited notable ACEI was assayed in SHR.
Section snippets
Production of fermented milk
Selected microorganisms were isolated from raw milk and were deposited in the Spanish Type Culture Collection (Colección Española de Cultivos Tipo, CECT). Fermented milk was prepared as previously described (Muguerza et al., 2006). Briefly, pre-cultures of E. faecalis strains CECT 5727, 5728, 5826 and/or 5827 were prepared in sterile reconstituted 10% (w/w) skimmed milk powder incubated overnight at 30 °C. A 3% v/v of corresponding pre-culture was added to reconstituted skimmed milk powder and
ACEI activity during milk fermentation
Figs. 1A and B illustrate, respectively, the bacterial growth, pH, and the proteolytic activity as measured by the OPA method during milk fermentation with the four selected strains of E. faecalis. Progress of the proteolysis was significant until the bacteria reached the stationary phase. After 24 h of fermentation, proteolysis still increased but at a lower rate. With regard to the development of ACEI activity during milk fermentation (Fig. 1C), a small increase was found during the first 6 h
Discussion
It is recognised that milk fermentation with lactic acid bacteria can generate a large number of peptides including some with potentially bioactive properties. However, the production of antihypertensive peptides by milk fermentation has not always been successful. Several authors have pointed out that milk fermentation with commercial starter strains cannot generate peptides with ACEI activity although these fermented products can act as precursors of ACEI peptides after hydrolysis with
Conclusions
Milk fermented with different strains of E. faecalis contained peptides with ACEI and antihypertensive activity. The protein fragments responsible for this activity were proline-rich peptides of low molecular mass. Of special interest is fragment β-CN f(133–138), LHLPLP, which exhibited potent antihypertensive activity at a dosage of just 2 mg kg−1 of body weight. It must be highlighted that the antihypertensive activity of this peptide is comparable to the activity found for the antihypertensive
Acknowledgments
The above-mentioned experiments were performed as authorised for scientific research (European Directive 86/609/CEE and Royal Decree 223/1988 of the Spanish Ministry of Agriculture, Fisheries and Food). This work has received financial support from the projects AGL2004-06903-C02-01 and FIT-06000-2002-30. A. Quirós was the recipient of a fellowship of the I3P Programme from the Consejo Superior de Investigaciones Científicas.
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