Release of Short and Proline-Rich Antihypertensive Peptides from Casein Hydrolysate with an Aspergillus oryzae Protease
Calpis Co., Ltd., Edo, Tokyo, Japan Journal of Dairy Science
(Impact Factor: 2.57).
11/2004; 87(10):3183-8. DOI: 10.3168/jds.S0022-0302(04)73453-0
Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages. Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.
Available from: Mohammad Mousavi
- "Therefore, changes in the protein structure induced by enzyme action or hydrolysis condition would presumably cause a decline in the generated or intrinsic antioxidative properties of the intact whey protein. Nevertheless , a further increase in the temperature promoted antioxidant capacity (Fig. 2) possibly due to the release of bioactive peptides or amino acids whose production were more apparent at central pH values likely to be the optimum pH of enzyme reaction (Mizuno et al. 2004). As expected from the above hypotheses, prolonged proteolysis at pH values far from the isoelectric point of whey proteins promoted the antioxidant activity (Fig. 3). "
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ABSTRACT: The main aim of the present study was to optimize the hydrolysis conditions of whey protein isolate digested by a protease preparation from Aspergilus oryzae through response surface method (RSM) in order to achieve the maximum angiotensin I-converting enzyme (ACE)-inhibitory activity and antioxidant properties. The effects of hydrolysis conditions including time (2, 13, 24, 35, and 46 h), temperature (40, 45, 50, 55, and 60 °C) and pH (6, 6.5, 7, 7.5, and 8) were investigated on the bioactivity of whey protein hydrolysates. Each process parameter emerged to have a dual effect on bioactivity; that is, increase in all variables promoted bioactive peptide generation through facilitating enzyme access to the primary protein sequence by partial unfolding of the compact globular assemblies of whey proteins. However, prolonged digestion at high temperatures and alkaline pH were concomitant with decreased bioactivity which are attributed to hydrolysate aggregation and splitting of bioactive peptides into biologically inactive counterparts, respectively. Nonetheless, some discrepancies were observed between the trend of ACE-inhibitory activity and that of antioxidant activity changes which was explained in light of their general characteristics. RSM efficiently identified the critical levels of each variable to obtain maximum bioactivity. It was shown that hydrolysate prepared at 56.54 °C and pH 6.04 resulting from digestion for 3.89 h exerted 74% ACE-inhibitory activity, 666.31 μM trolox equivalent/mg antioxidant activity, and 14.03% hydrolysis degree.
Available from: Mercedes Ramos
- "The first fermented milk with antihypertensive activity was produced with a combination of Lactobacillus helveticus and Saccharomyces cerevisiae and contained two ACE-inhibitory tripeptides , isoleucine–proline–proline (IPP) and valine–proline–proline (VPP) (Nakamura et al., 1995). These two tripeptides have also been produced by milk fermentation with Lactobacillus helveticus LBK-16H (Seppo et al., 2003) and casein hydrolysis with a protease from Aspergillus oryzae (Mizuno et al., 2004). These tripeptides exhibited potent ACE-inhibitory activity and proven antihypertensive effects in both animals and humans (Sipola et al., 2002; Hata et al., 1996). "
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ABSTRACT: The Lowpept is a powdered casein hydrolysate containing the antihypertensive peptides RYLGY and AYFYPEL, two sequences that correspond to alpha(s1)-casein f (90-94) (RYLGY) and alpha(s1)-casein f (143-149) (AYFYPEL). To support the safety, Lowpept has been examined in an acute and in a 4-week repeated dose oral toxicity studies in rats. Powdered casein hydrolysate administered in a single oral gavage dose of 2000 mg/kg resulted in no adverse events or mortality. Also, casein hydrolysate administered as a daily dose of 1000 mg/kg for 4 weeks by gavage resulted in no adverse events or mortality. No evidence or treatment-related toxicity was detected during both studies. Data analysis of body weight gain, food consumption, clinical observations, blood biochemical, haematology, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. It is concluded that the casein hydrolysate containing the peptides RYLGY and AYFYPEL orally administered to rats was safe and that not treatment-related toxicity was detected even at the highest doses investigated in both acute (2000 mg/kg of body weight) and repeated dose (4 weeks) oral (1000 mg/kg of body weight) toxicity studies.
Available from: onlinelibrary.wiley.com
- "Studies of physiologically functional peptides obtained from food have greatly progressed recently. For instance, lactotripeptide (Nakamura et al. 1995; Seppo et al. 2003; Mizuno et al. 2004; Sano et al. 2005) is the main focus of these investigations. These peptides are composed of several amino acids, and they may be produced by the combined reaction of several proteases. "
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ABSTRACT: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties.
The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1).
Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding.
PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.
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