Jean-Luc Gaillard

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine αS2-casein

Angiotensin‐I‐converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine αS2‐casein (αS2‐CN) was extensively investigated. Forty‐three peptide peaks were isolated and tested. Seven casokinins (i.e. CN‐derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 μM. Peptides αS2‐CN (f174–181) and αS2‐CN (f174–179) had IC50 values of 4 μM. Surprisingly, deletion of the C‐terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.

Tryptic hydrolysis of bovine αS2-casein: identification and release kinetics of peptides

Abstract Bovine α S2 -casein was purified by anionic exchange and hydrophobic interaction chromatographies. The purified α S2 -casein was hydrolysed with trypsin; the resulting peptides were identified by amino acids composition and on line reversed-phase high performance liquid chromatography coupled with electrospray mass spectrometry. A study on the kinetics of peptide release enabled discrimination between different protein areas according to their surface-accessibility. Data from the kinetic study were reconciled with secondary structure predictions and a hypothetical model of the structural organisation of purified bovine α S2 -casein in solution was proposed.

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Abstract.Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa.

Inhibitory effect of αS1- and αS2-casein hydrolysates on angiotensin I-converting enzyme in human endothelial cells in vitro, rat aortic tissue ex vivo, and renovascular hypertensive rats in vivo

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from alpha(S1)- and alpha(S2)-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood.

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two‐dimensional electrophoresis (2‐DE) protein profile was obtained from three independent analyses of 2‐DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2‐DE profile after silver staining. These preliminary results contribute to the setting up of a two‐dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42°C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2‐DE silver‐stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2‐D PAGE markers of pI and Mr for S. thermophilus.

In vitro digestibility of α-casozepine, a benzodiazepine-like peptide from bovine casein, and biological activity of its main proteolytic fragment.

α-Casozepine is a peptide, corresponding to the sequence 91-100 of the bovine α(s1)-casein, displaying anxiolytic activity in the rat. The α(s1)-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91-97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.

Improvement of a method for the measurement of lactoperoxidase activity in milk

Abstract Lactoperoxidase (LPO) is one of the two most used indigenous enzymes to evaluate milk heat treatment, alkaline phosphatase being the other. In the present work, LPO activity was measured using Clarifying Reagent ® to facilitate spectrophotometric measurement in milk. Satisfying repeatability was obtained with a CV of 5.2% ( n =33). Results obtained by this method correlated well with the classic Rothenfusser method ( R 2 =0.97; n =20) and also with another method using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate diammonium salt) ( R 2 =0.98; n =21). This very easy rapid colorimetric method avoided preliminary casein precipitation and filtration steps. It has been shown to be suitable for routine characterisation of heat treatment of milk.

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism,1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional1H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 310-helicoid structure in which the E6...R10 ionic bridge stabilizes the C-terminus.

SPECIFIC INTERACTION BETWEEN ANIONIC PHOSPHOLIPIDS AND MILK BOVINE COMPONENT PP3 AND ITS 119-135 C-TERMINAL FRAGMENT

Abstract The behaviour of component PP3, a bovine milk protein with emulsifying properties, was investigated at the air–water interface and in a lipidic environment using the monolayer technique. The amphipathic 119–135 C-terminal fragment of PP3 was also tested since we proposed, on the basis of structural analysis, that this region is probably responsible for the surface-active properties of the protein. This hypothesis was confirmed by the tensiometric measurements at the air–water interface in which the addition of the C-terminal peptide increased the surface pressure with a similar amplitude as the whole protein. Penetration measurements into lipidic monolayers indicated that the insertion of component PP3 and its C-terminal peptide is the highest with anionic phospholipids in a gel state. Moreover, the electrostatic attractions provided by anionic phospholipids are essential for the peptide interaction. We also showed by Fourier transform infrared spectra study, that the peptide displays a β-type conformational state in aqueous solution and in the presence of solvant or anionic phospholipid (DPPG). In contrast, the protein adopts in aqueous solution an α helical conformation which remains the dominant conformational state in the presence of DPPG although the apparition of β-structure is detected.