Plastein from hydrolysates of porcine hemoglobin and meat using Alcalase and papain.

Plastein is defined as a protease-induced peptide aggregate and has been explored for over a century. This study investigated the effects of Alcalase and papain on plastein formation in protein hydrolysates of porcine hemoglobin and meat by measuring turbidity, particle size distribution, free amino groups and chemical interactions, as well as identifying the soluble peptides remaining in solution by LC-MS/MS. The results showed that Alcalase induced more peptide aggregation than papain in terms of increases in turbidity and particle size. Porcine hemoglobin was better than meat in inducing plastein formation in a short reaction time. Besides, covalent bonds involving peptide bonds and disulfide bonds were not crucial in the plastein reaction, instead a high proportion of hydrophobic interactions dominated the plastein. Not all peptides of both hydrolysates took part in plastein formation, and the regions of sequence that were prone to aggregation were visualized by Peptigram.

The influence of pH, protein concentration and calcium ratio on the formation and structure of nanotubes from partially hydrolyzed bovine α-lactalbumin.

Formation of nanotubes from partially hydrolysed α-lactalbumin (α-La) was investigated at five pH values, two concentrations of α-La and two calcium levels. Nanotubes were formed under almost all combinations of the investigated factors, and for the first time the formation of nanotubes at low pH (4.0) and low protein concentration (10 g l-1) was observed. Only one sample (10 g l-1, calcium ratio 2.4, and pH 7.5) formed mainly fibrils instead of nanotubes. By altering the three investigated factors, fibrils and/or aggregates were sometimes formed together with nanotubes resulting in transparent, semi-transparent, or non-transparent gels, or sediments. However, structural modelling based on small-angle X-ray scattering data indicated that the formed nanotubes were only to a minor degree affected by the investigated factors. The majority of the nanotubes were found to have an outer diameter of around 19 nm, an inner diameter of 6.6 nm and a wall thickness of 6.0 nm, except for three samples at low α-La concentrations and high calcium levels which exhibited slightly smaller dimensions. These three factors affected the hydrolysis as well as the self-assembly rate, resulting in the observed differences. However, these factors did not influence the architecture of the self-assembled nanotubes, and the lateral spacing of the individual parallel ß-sheet motifs was found to be 1.05 ± 0. 03 nm for all nanotubes. This study provides novel fundamental knowledge of the formation and structure of α-La nanotubes under different conditions, which will facilitate future application of these nanotubes in food and pharmaceutical areas.

Antioxidant peptides (<3 kDa) identified on hard cow milk cheese with rennet from different origin.

The effect of the addition of rennet from different origin (animal, plant and microbial) on the release of potential antioxidant peptides was evaluated in hard cow milk cheese. After cheese making, chemical parameters, colour, TBARs and riboflavin content were determined in the final product. Antioxidant activity of the water soluble extracts from cheese was evaluated by the DPPH radical scavenging and metal chelating activity assays. Separation of peptide fractions (<3 kDa) was performed by RP-HPLC and fractions with potential antioxidant activity were analyzed by LC-MS/MS for further identification of peptides. Results suggested that colour and riboflavin content were affected by the type of rennet used (P < .05). On the other hand, extracts of cheese with microbial rennet showed the highest antioxidant activity (P < .05). Only nine fractions isolated from the extracts showed either DPPH radical scavenging or metal chelating activities. Peptides EIVPN and DKIHPF, previously reported in other studies by showing ACE-inhibitory activity, were identified in these fractions and revealed high metal chelating activity. Moreover, a new peptide, with also high metal chelating activity, has been identified (VAPFPQ). In conclusion, it would be worthwhile to synthesis these peptides and confirm their antioxidant and metal chelating activities.

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.

Antioxidant capacity of hydrolyzed animal by-products and relation to amino acid composition and peptide size distribution.

The antioxidative capacity of six different tissue hydrolysates (porcine colon, heart and neck and bovine lung, kidney and pancreas) were tested by three different assays monitoring iron chelation, ABTS radical scavenging and inhibition of lipid oxidation in emulsions, respectively. The hydrolysates were also investigated with respect to amino acid composition and peptide size distribution. The hydrolysates contained peptides ranging from 20 kDa to below 100 Da with a predominance of peptides with low molecular weight (53.8 to 89.0 % below 3 kDa). All hydrolysates exhibited antioxidant activity as assessed with all three methods; inhibition of lipid oxidation ranging from 72 to 88 % (at a final protein concentration of 7 mg/mL), iron chelation capacity from 23 to 63 % (at 1.1 mg/mL), and ABTS radical scavenging from 38 to 50 % (at 10 µg /mL). The antioxidant activity did not correlate with the proportion of low molecular weight peptides in the hydrolysed tissues, but with the content of specific amino acid residues. The ABTS radical scavenging capacity of the tissues was found to correlate with the content of Trp, Tyr, Met and Arg, whereas the ability to inhibit the oxidation of lineoleic acid correlated with the content of Glu and His. The chosen animal by-products thus represent a natural source of antioxidants with potential for food application.

Dissociation and reduction of covalent ß-lactoglobulin-quinone adducts by dithiothreitol, tris(2-carboxyethyl)phosphine, or sodium sulfite.

Covalent protein-phenol adducts, generated by reaction of protein nucleophiles with quinones, have recently attracted increased attention because the interactions change the functionality and physicochemical properties of proteins in biological and food systems. The formation of such covalent adducts between ß-lactoglobulin (ß-LG) and the quinone of 4-methylcatechol, 4-methylbenzoquinone (4MBQ), and subsequent reduction by dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or sodium sulfite was investigated by mass spectrometry. The results showed that 19.0 ± 8.8% of ß-LG reacted with 4MBQ when present in equimolar ratio at 20°C (pH 8.0) to yield the protein-phenol adduct (ß-LG-Q). Following treatment with sulfite, DTT, or TCEP, 75, 68, or 36%, respectively, of the formed ß-LG-Q adduct dissociated. Different reaction mechanisms were proposed for the reduction of ß-LG and ß-LG-Q by each of the reducing agents. These results show that on reductive sample preparation for analysis of protein samples, not only are protein polymers formed through oxidative disulfide bonds reduced into the individual protein constituents but also a large part of any protein-phenol adducts present will dissociate and, thus, give a false picture of the level of protein-protein interactions that have occurred in the sample.

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.

Antioxidant capacity of hydrolyzed porcine tissues.

The antioxidative capacity of seven different porcine tissue hydrolysates (colon, appendix, rectum, pancreas, heart, liver, and lung) were tested by four different assays, including iron chelation, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) radical scavenging, and inhibition of lipid oxidation. All hydrolyzed tissues displayed antioxidant capacity in all four assays, with colon, liver, and appendix as the three most potent inhibitors of lipid oxidation (47, 29, and 27 mmol/L trolox equivalent antioxidant capacity [TEAC], respectively) and liver, colon, pancreas, and appendix as the four most potent iron chelators (92% ± 1.1, 79.3% ± 3.2, 77.1% ± 1.8, and 77% ± 2.3, respectively). Furthermore, colon and appendix showed good radical scavenging capacities with ABTS scavenging of 86.4% ± 2.1 and 84.4% ± 2.9 and DPPH scavenging of 17.6% ± 0.3 and 17.1% ± 0.2, respectively. Our results provide new knowledge about the antioxidant capacity of a variety of animal by-products, which can be transformed into antioxidant hydrolysates, thereby creating added value.

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.

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.

Self-assembly of partially hydrolysed alpha-lactalbumin.

Hydrolysis of the whey protein alpha-lactalbumin with a specific serine protease has been shown to result in regular nanotubes of approximately 20 nm in outer diameter and reaching several mum in length. Tubular assembly depends on the concentration of protein as this determines how far the hydrolysis proceeds. A concentration of 30 g L(-1) is a prerequisite for tubular formation, as is a minimum concentration of calcium. At lower protein concentrations calcium-independent formation of linear fibrils (approximately 5 nm in diameter) is favoured. Possible applications of alpha-lactalbumin nanotubes include use as a viscosifier and gelling agent and also pharmaceutical utilization (such as targeted drug release) and use in nanotechnology can be envisioned.

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.

Influence of calcium on the self-assembly of partially hydrolyzed alpha-lactalbumin.

Self-assembly of alpha-lactalbumin after partial hydrolysis by a protease from Bacillus licheniformis can result in nanotubular structures, which show many similarities to microtubules. Calcium plays a crucial role in this process. The objective of this investigation was to study the role of calcium in more detail. The kinetics of the hydrolysis step and the self-assembly step were monitored by respectively liquid chromatography-mass spectrometry and dynamic light scattering. The microstructure of the gels finally formed was investigated by transmission electron microscopy. This investigation demonstrates that calcium accelerated the kinetics of the self-assembly, but it had no effect on the hydrolysis kinetics. As a result of the accelerated self-assembly kinetics at a high calcium concentration, the time of gelation decreased as well. A minimum concentration of calcium needed to obtain the tubular alpha-lactalbumin structures was determined. Below R = 1.5 (mole calcium/mole alpha-lactalbumin), turbid gels with randomlike structure were obtained. Between R = 1.5 and R = 6, translucent gels with a fine stranded network of tubules were formed, while higher calcium concentrations had a negative effect on the tubule formation, resulting in amorphous structures. The optimum calcium concentration for alpha-lactalbumin nanotube formation seemed to be around R = 3.

Protease-induced aggregation of bovine alpha-lactalbumin: identification of the primary associating fragment.

Details in the hydrolysis of alpha-lactalbumin known to result in formation of highly ordered nanotubules, was investigated by incubation of solutions with 10 g alpha-lactalbumin/l with a specific protease from Bacillus licheniformis (BLP). After 50 min of incubation, soluble aggregates were formed, the concentration of which increased until precipitation occurred after 200 min. The latter aggregates were dissolved in urea or at low pH, like the nanotubules characteristic of gels formed by the action of BLP on alpha-lactalbumin at 100 g/l. On the molecular level, alpha-lactalbumin was initially cleaved into two large hydrophobic fragments with masses of 11.6 and 11.3 kDa, which in turn were cleaved in a stepwise manner into the ultimate fragment of 8.8 kDa. This fragment was the predominating component in the insoluble aggregates, and was identified as the sequences 26-37 and 50-113 of alpha-lactalbumin linked together by a disulphide bond. Cleavage of alpha-lactalbumin into this fragment probably created new hydrophobic surfaces and new calcium binding sites allowing its association into ordered structures.