Jeanette Otte

Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates

In development of fermented dairy products and protein hydrolysates with high inhibitory activity towards angiotensin-converting enzyme (ACE), it is crucial to have a reliable assay for measuring the ACE activity. In the present study, the performance of two commonly used assays based on synthetic N-derivates of tripeptides as substrates were tested with respect to reliability in determination of ACE activity per se and to the inhibitory effect of a tryptic whey protein digest and captopril. In one test, the ACE activity was calculated from the amount of hippuric acid liberated from hippuryl-His-Leu (HHL) during 30 min of incubation with ACE, as quantified after HPLC separation of reaction products. In the other assay, the ACE activity was measured directly as the rate of decrease in the absorbance at 340 nm during the first 30 min of ACE catalysed hydrolysis of furanacroloyl-Phe-Glu-Glu (FA-PGG). Both assays, in the absence of inhibitor, showed a good performance with relative standard deviation between replicate samples around 7%. In the presence of inhibitor solutions, relative standard deviations for both assays varied between 1 and 18% for the variously diluted inhibitors. Both assays gave values for the concentration of inhibitor needed to inhibit ACE by 50% similar to those previously reported for whey protein digests and captopril. Different results from the two assays, however, emphasize the importance of controlling the actual ACE-activity for comparison between assays. The limitations of each assay are discussed. Considering the fewer steps in the assay using FA-PGG as substrate, and thus less time and chemicals consumed per sample, and the simpler equipment needed, this assay is recommended for the screening of clear peptide samples.

Emulsifying Properties of Milk Proteins Cross-linked with Microbial Transglutaminase

Oil-in-water emulsions containing 20% oil were prepared with n-tetradecane and sodium caseinate or β-lactoglobulin in the aqueous phase. The proteins had been cross-linked with microbial transglutaminase prior to emulsification or were cross-linked in the emulsions after emulsification. Extensive cross-linking reduced the stability of the emulsions to coalescence or strong flocculation, as determined by droplet size measurements, whereas limited cross-linking improved coalescence stability. The creaming stability of milk protein stabilized emulsions was improved even with relatively extensive cross-linking and this was ascribed to changes in the adsorbed layer or increased viscosity of the continuous phase. Cross-linking greatly reduced Ostwald ripening or aggregation of the milk protein stabilized emulsions in the presence of ethanol, probably due to a reduction of the diffusion of oil from the emulsion droplets because of increased viscosity/elasticity of the adsorbed layer or due to hindering of the aggregation of protein, that in the non-cross-linked state probably results in a collapse of the surface protein layer.

Molecular self-assembly of partially hydrolysed α-lactalbumin resulting in strong gels with a novel microstructure

Gelation of alpha-lactalbumin (alpha-la) incubated with a protease from Bacillus licheniformis (BLP) at 50 degrees C for 4 h was monitored using small oscillatory shear and the large deformation properties of final gels were characterized by uniaxial compression. Transmission electron microscopy was used to visualize the microstructure. Gels made from alpha-la (10 g/l) using BLP were almost transparent, although somewhat whitish, and they were more than 20 times stiffer (measured as complex modulus) than equivalent gels made from beta-lactoglobulin (beta-lg) at the same concentration. The microstructure of the gels consisted of non-branching, apparently hollow strands with a uniform diameter close to 20 nm, similar in overall structure to microtubules. Adding Ca2+ in amounts of 50 or 100 mM changed the spatial distribution of the strands and resulted in a reduction in the failure stress recorded in uniaxial compression. Apart from affecting the microstructure, Ca2+ was shown to be essential for the formation of the gels. It is proposed. that the mechanism behind the self-assembly of the partially hydrolysed alpha-la into long tubes is a spatially restricted creation of ionic bonds between Ca2+ and carboxyl acid groups on peptide fragments resulting from the action of BLP on alpha-la. Proteolysis of alpha-la with BLP in the presence of Ca2+ thus results in formation of a strong gel with a microstructure not previously observed in food protein systems.

Effect of limited hydrolysis on the interfacial rheology and foaming properties of β-lactoglobulin A

Hydrolysis of beta-lactoglobulin (beta-Lg), genetic variant A, using a serine protease specific for glutamic and aspartic acid residues from Bacillus licheniformis (BLP), resulted in improved foam overrun and foam stability. Limited hydrolysis (19-26% hydrolysed beta-Lg) led to a more rapid increase in the viscoelastic properties of air/water interfacial films and a concomitant increase in foam overrun compared with intact beta-Lg, presumably due to increased exposure of hydrophobic areas. The increased exposure did not, however, cause formation of an interfacial layer with increased viscoelastic properties. More extended hydrolysis (86% hydrolysed beta-Lg) resulted in a higher initial overrun than the unhydrolysed sample and the best foam stability. The interfacial elasticity and viscosity, though, was the lowest observed. Thus, high maximum values of these interfacial properties are not necessary prerequisites for formation of a voluminous and stable foam.

Enzyme-induced gelation of whey proteins: Effect of protein denaturation

Abstract The enzyme-induced gelation properties of unheated and denatured whey proteins (WP) were investigated. Solutions (9%, w v ) of whey protein isolate (WPI) were denatured by heating at 80 °C for 2–30 min, and gelation was induced by incubation with a Bacillus licheniformis protease (40 °C, pH 7.0). The gelation characteristics, as well as gel strength and microstructure, were examined. The heat treatments resulted in irreversible denaturation of up to 98% of the WP. Bacillus licheniformis protease (BLP) was able to induce gelation of both unheated and pre-heated WP, but the gelation process was strongly dependent on the extent of denaturation (%D). Higher %D resulted in earlier gelation and a faster increase in gel firmness. The relation between %D and the rate of gel firming was almost linear. The strength of the enzyme-induced gels increased continuously during 30 h of incubation. After 9 h, the gel with 98%D was 3.2 times stronger than that with 47%D, and 18 times stronger than the gel from unheated WPI (≈8%D). The latter gel had a particulate microstructure, whereas the gel from the highly denatured WPI (98%D) had a fine-stranded structure. This enzyme-induced gelation of partly denatured WP may be of interest for food applications.

Bioactive peptides from caseins released by cold active proteolytic enzymes from Arsukibacterium ikkense.

Proteolytic enzymes secreted by the cold-adapted microorganism Arsukibacterium ikkense were tested for their ability to degrade caseins at low temperature and produce bioactive peptides. The caseins were extensively degraded (90%) after 24h of hydrolysis at 5°C and completely degraded at 25°C, and many novel peptides were formed. The most hydrolysed sample showed high angiotensin I converting enzyme (ACE)-inhibitory and antioxidant activity, and a number of potent ACE-inhibitory and antioxidant peptides were identified. The presence of tyrosine seemed fundamental for both ACE-inhibitory and antioxidant activity, while phenylalanine seemed to potentiate the antioxidant activity. The novel peptide YPELF was found to have strong radical scavenging and lipid oxidation inhibitory activities, with IC50 for both around 3.5μM. None of the hydrolysates showed antimicrobial activity. Secreted enzymes from cultures of A. ikkense could thus be a valuable enzyme preparation for inexpensive, energy-efficient production of potent bioactive peptides from caseins in milk at low temperatures.

Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides

Bovine collagen was pre-treated (boiled or high pressure (HP)-treated) and then hydrolysed by 6 proteases. The degree of hydrolysis (DH) and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates were measured. All enzymes used were able to partly degrade collagen and release ACE-inhibitory peptides. The highest ACE-inhibitory activity was obtained with Alcalase. Pretreatment significantly influenced the DH and ACE-inhibition. For most enzymes, boiling for 5 min resulted in a significantly higher DH and ACE-inhibitory activity. With Alcalase and collagenase, hydrolysis and release of ACE-inhibitory peptides occurred without any pretreatment, but HP-treatment significantly improved the DH and ACE-inhibitory activity. HP did not markedly affect the hydrolysis with the other enzymes. The major peptides obtained with Alcalase were identified; all were released from the triple helix structure of collagen. Many of these peptides had C-terminal sequences similar to known ACE-inhibitory peptides. The present results suggest that collagen-rich food materials are good substrates for the release of potent ACE-inhibitory peptides, when proper pre-treatment and enzymatic treatment is applied.

Isolation and identification of low molecular weight antioxidant compounds from fermented "chorizo" sausages.

This work is focused on the determination of low molecular weight compounds extracted from samples of fermented sausages. The antioxidant activity of fractions isolated from chorizo extracts was tested by their ability to quench free radicals by the DPPH-radical scavenging assay. Natural dipeptides and metabolites characteristic of meat were abundant in the fractions isolated by RP-HPLC from chorizo extracts. Due to extensive degradation during the ripening of chorizo, the extracts did not contain many peptides in a concentration that allowed identification. However, many free amino acids were identified by LC-MS/MS and HILIC-MS/MS. The fractions with the most hydrophilic compounds showed the highest antioxidant activity.

Antioxidant activity of cod (Gadus morhua) protein hydrolysates: Fractionation and characterisation of peptide fractions.

This study aimed to characterise peptide fractions (>5kDa, 3-5kDa and <3kDa) with antioxidative activity obtained from a cod protein hydrolysate. The free amino acids in all fractions were dominated by Ala, Gly, Glu and Ser. The total amino acid composition had high proportions of Lys, Ala and Glu. The 3-5kDa and <3kDa fractions were further fractionated by size exclusion chromatography. All sub-fractions showed high Fe(2+) chelating activity. The DPPH radical-scavenging activity of the 3-5kDa fraction was exerted mainly by one sub-fraction dominated by peptides with masses below 600Da. The DPPH radical-scavenging activity of the <3kDa fraction was exerted by sub-fractions with low molecular weight. The highest reducing power was found in a sub-fraction containing peptides rich in Arg, Tyr and Phe. Both free amino acids and low molecular weight peptides thus seemed to contribute to the antioxidative activity of the peptide fractions, and Tyr seemed to play a major role in the antioxidant activity.

High pressure treatment of brine enhanced pork affects endopeptidase activity, protein solubility, and peptide formation

In order to study the effect of high-pressure (HP) treatment and two different methods of brine addition (important for lysosomal membrane destabilisation) on lysosomal enzymes activity and protein degradation, pork semitendinosus muscle was brine enhanced by injection or tumbling, and HP treated at 600 MPa following storage at 2 °C for up to 8 weeks. In this report a novel protocol for SDS gelatin zymography was established, and an increase of cathepsin B and L activity after HP treatment was shown followed by a decrease during storage. No calpain activity was detected following HP treatment. HP treatment was shown to induce a decrease in protein solubility in both myofibrillar and sarcoplasmic fractions. LC-MS analysis of these fractions showed changes in the peptide pattern during storage. Western blot analysis showed that troponin-T was indeed degraded during storage after HP treatment. The results therefore suggest that HP treatment induced an increase in cathepsin activity, which subsequently affected the myofibrillar protein degradation pattern in pork meat.