Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

ACE-inhibitory activity of probiotic yoghurt

In this study, the in vitro angiotensin-converting enzyme (ACE)-inhibitory (ACE-I) activity of peptide fractions from different yoghurt batches was assessed. Inhibition of ACE activity resulted in an overall antihypertensive effect. Yoghurts were prepared either using a sole yoghurt culture including Lactobacillus delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342, or L. acidophilus L10, L. casei L26 and Bifidobacterium lactis B94 in addition to yoghurt culture. ACE-I activity was determined at weekly intervals during 28 days of cold storage. Peptide fractions showing high ACE-I activity were further purified using multiple-steps of RP-HPLC. All probiotic yoghurts showed appreciable ACE-I activity during initial stages of storage compared with the control yoghurt, with a significant (p<0.05) decrease afterwards. The ACE-I activity ranged from IC₅₀ of 103.30–27.79 μg mL⁻¹ with the greatest ACE inhibition achieved during first and third week of storage. The in vitro ACE-I activity could be related to the peptide liberation via degradation of caseins. In total, 8 ACE-I peptides were characterized originating from α_s2-casein (1), κ-casein (2) and β-casein, of which two well-known ACE-inhibiting peptides, namely Val–Pro–Pro (VPP) and Ile–Pro–Pro (IPP), were identified. These peptides are already used in commercial products.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC₅₀ of 67.2 ± 1.8 μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC₅₀ of 19.3 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.     

Formation of whey protein/κ-casein complexes in heated milk: Preferential reaction of whey protein with κ-casein in the casein micelles

Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90 °C 10 min⁻¹) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles.     

Isolation of antioxidative and ACE inhibitory peptides from protein hydrolysate of skipjack (Katsuwana pelamis) roe

Bioactive peptides from protein hydrolysate of defatted skipjack (Katsuwonus pelamis) roe with 5% degree of hydrolysis (DH) prepared by Alcalase digestion were isolated and characterised. Two active fractions with ABTS radical scavenging activity (973.01–1497.53 μmol TE/mg sample) and chelating activity (0.05–0.07 μmol EE/mg sample) from consecutive purification steps including ultrafiltration, cation exchange column chromatography and reverse phase high performance liquid chromatography (RP-HPLC), were subjected to analysis of amino acid sequence by LC–MS/MS. Seven dominant peptides with 6–11 amino acid residues were identified as DWMKGQ, MLVFAV, MCYPAST, FVSACSVAG, LADGVAAPA, YVNDAATLLPR and DLDLRKDLYAN. These peptides were synthesised and analysed for ACE-inhibitory activity and antioxidative activities. MLVFAV exhibited the highest ACE inhibitory activity (IC₅₀ = 3.07 μM) (p < 0.05) with no antioxidative property, whilst DLDLRKDLYAN showed the highest metal chelating activity, ABTS radical and singlet oxygen scavenging activities. Therefore, peptides prepared from skipjack roe could be further employed as a functional food ingredient.     

Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions

In this study, rapeseed protein isolate was hydrolyzed with various proteases to obtain hydrolysates that were separated by membrane ultrafiltration into four molecular size fractions (<1, 1–3, 3–5, and 5–10 kDa). Alcalase hydrolysis significantly (p < 0.05) produced the highest yield of protein hydrolysate while Flavourzyme produced the least. The <1 kDa fraction was the most abundant after the membrane ultrafiltration of the protein hydrolysates, which indicates that the proteases were efficient at reducing the native rapeseed proteins into low molecular weight peptides. Antioxidant properties of the resulting hydrolysates and membrane fractions were characterized and results showed the Pepsin + Pancreatin (P + P) protein hydrolysate had significantly highest (p < 0.05) scavenging activity against DPPH radical among the unfractionated enzymatic hydrolysates. But the P + P hydrolysate was not as effective as other hydrolysates during long-term inhibition of linoleic acid oxidation. For most of the samples, fractionation into the <1 kDa peptides significantly (p < 0.05) improved DPPH and superoxide scavenging properties when compared to the unfractionated protein hydrolysates. Only the <1 kDa fraction showed ferric reducing antioxidant power and the effect was dose-dependent. Overall, Alcalase and Proteinase K seem to be more efficient proteases to release antioxidant peptides from rapeseed proteins when compared to P + P, Flavourzyme and Thermolysin.     

Comparison of subjective grading and objective assessment in meibography

AimTo analyse repeatability of subjective grading and objective assessment in non-contact infra-red meibography.MethodsMeibography photographs of 24 subjects (female 14; mean age = 46; range = 19–69 years, upper-lid images = 12, lower-lid images = 12) were classified in two sessions by three experienced observers (OI, OII, OIII). Relative area or portion affected by meibomian glands (MG) loss was classified applying three different grading scales in randomized order: a four-grade scale (4S) (degree 0 = no partial glands; 1 = <25% partial glands; 3 = 25–75% partial glands; 3 = >75% partial glands), a pictorial five-grade scale (5S) (degree 0 = no meibomian gland loss (MGL); 1 = <25% MGL; 3 = 26–50% MGL; 3 = 51–75%; 4 = >75% MGL) and objectively by a 100-grade scale (DA) applying ImageJ software.ResultsObserved MG loss ranged from 0% to 69%. Intra-observer agreement of the 5S (OI: κ = 0.80, p < 0.001; OII: κ = 0.40, p = 0.009; OIII κ = 0.81, p < 0.001) was better than of the 4S (OI: κ = 0.79, p < 0.001; OII: κ = 0.15, p = 0.342; OIII κ = 0.50, p = 0.0071). Intra-observer agreement of OI and OIII (±0.88 (95% confidence interval), ±1.305) was better than of OII (±2.21) in 4S and 5S (±0.99, ±2.00 and ±0.91; OI, OII and OIII, respectively) while it was relatively similar in DA (±18, ±17 and ±17). Inter-observer agreement was better in DA (OI–OII: ±13, OI–OII: ±19, OII–OIII: ±26) than in 4S (OI–OII: ±1.76; OI–OIII: ±1.29 and OII–OIII: ±1.31) or 5S (OI–OII: ±1.49; OI–OIII: ±0.91 and OII–OIII: ±1.20).ConclusionIntra-observer and inter-observer agreement was better in computerized grading followed by the subjective five-grade scale and four-grade scale.     

Angiotensin-I converting enzyme (ACE) inhibitory tripeptides from rice protein hydrolysate: Purification and characterization

In this study, angiotensin-I converting enzyme (ACE) inhibitory tripeptides from rice protein hydrolysate were purified and their properties were further characterized. Val-Asn-Pro (VNP) and Val-Trp-Pro (VWP) were successfully purified from rice protein hydrolysate through multi-step isolation and purification comprising of ultrafiltration and DA201-C macroporous adsorption resin, followed by a two-step reverse phase high performance liquid chromatography (RP-HPLC). The sequences of tripeptides were separately assayed by using automated protein/peptide sequencer. In addition, the stability of the tripeptides against ACE and simulated gastrointestinal proteases, ACE-inhibitory kinetics, and their antihypertensive effects in vivo were evaluated. Our results showed that VNP (IC₅₀ value of 6.4 μM) and VWP (IC₅₀ value of 4.5 μM) were both competitive ACE inhibitors, and stability against ACE and gastrointestinal proteases of pepsin and chymotrypsin. Furthermore, single oral administration of the tripeptides VNP and VWP at 5 mg/kg body weight in spontaneously hypertensive rats (SHRs) significantly decreased the systolic blood pressure (SBP) of 29 and 38 mmHg (P < 0.05), respectively, and the antihypertensive effects of tripeptides could last for 8 h minimally. The identified unique characterization of VNP and VWP may enable the application of rice protein hydrolysate as functional foods or pharmaceuticals against hypertension.     

Interactions Leading to Formation of Casein Submicelles

Gel chromatography was employed in studies of interactions of soluble whole casein that was prepared by dissociation of casein micelles with ethylenedia-minetetraacetate. With increasing protein concentration at pH 6.6 and 37°C, components of whole casein associate to polymers that approach molecular radii with apparent upper limit of 9.4 ± .4 nm. With decreasing protein concentration, κ-casein dissociates from the other casein components. This was shown by analysis of the eluted protein boundary by gel electrophoresis and radial immunodiffusion. The peak maximum elution volume and the broad, skewed character of the separated κ-casein peak indicate that in whole casein κ-casein exists in a size distribution of disulfide bonded polymers. This apparently suggests that SH-κ-casein monomers aggregate independently of the other casein components in the growth of casein submicelles, and additional studies with the purified casein components support this concept. However, after disulfide bond reduction with dithiothreitol, chromatography of whole casein over the same concentration range did not result in separation of SH-κ-casein polymers, because all of the casein was eluted under one peak. These findings show that, in vivo, casein submicelles could be formed by interaction of SH-κ-casein monomers with those of α_s- and β-casein, followed by S-S-κ-casein polymer formation through oxidation after milking.     

The effect of ultra high-pressure homogenization (UHPH) on rennet coagulation properties of unheated and heated fresh skimmed milk

The effect of ultra-high pressure homogenization at a pressure of 179 MPa on the renneting of milk has been studied. The homogenization has a small effect on the diameters of casein micelles, because of the loss of some of their surface κ-casein. This modification of the structure leads to a slightly decreased rennet coagulation time. Interactions between the casein micelles in homogenized and unhomogenized milk samples started at a degree of proteolysis of the κ-casein of about 65–70%, although aggregation of the micelles did not start until over 90% proteolysis. Homogenization improved the coagulation properties of heated milk only slightly; however, it was shown that the removal of stabilizing repulsions between the casein micelles in the heated milk seemed to proceed in the same way as in unheated milk. The removal of the κ-casein has the same effect in heated and unheated milk samples, and the casein micelles are destabilized; it is only in the final aggregation step that the two milks differ.     

Protein interactions in milk protein concentrate powders

Solutions (5%, w/w, pH 6.9), made from six different pilot-plant milk protein concentrate (MPC) powders, were centrifuged (700 g for 10 min at 20 °C) to isolate the insoluble material. The sediment (insoluble material) was characterized using one-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM). The levels of solubility in MPC powders varied between 32% and 98%. The TEM results showed that the insoluble material consisted of large particles (∼100 μm) where the casein micelles are fused together by some form of protein–protein interactions. The 1D PAGE results showed that the amounts of insoluble material in MPC powders increased with storage time at elevated temperatures. This material consisted predominantly of α- and β-caseins. This material was formed largely by weak non-covalent (hydrophobic) protein–protein interactions that were dissociable under SDS-PAGE conditions. The 2D PAGE results demonstrated that the disulphide-linked protein aggregates present in MPC powders consisted of κ-casein, β-lactoglobulin and some α_s2-casein, and not all of this material was sedimented. These aggregates were not considered to play a major role in the formation of the insoluble material.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Structure–mechanism relationship of antioxidant and ACE I inhibitory peptides from wheat gluten hydrolysate fractionated by pH

The aims of this study were to assess bioactive properties (ACE inhibition and antioxidant capacity) from wheat gluten hydrolysate peptides fractionated by pH (4.0, 6.0 and 9.0), to determine peptide action mechanism, and to relate it to the secondary structure and functional groups of peptides. Gluten hydrolysate extracts (GHE) were enriched in peptides with medium hydrophobicity and molecular weight (≈ 60% MH and 5.5 kDa, respectively). Gluten peptides inhibited ACE I by uncompetitive mechanism and a direct relationship between α-helix structure and IC50% value was obtained (r = 0.9127). TEAC and cooper chelating activity from GHE 6.5 were the highest and directly correlated with MH peptides. GHE 9.0 had high carotene bleaching inhibition (47.5 ± 0.3%) and reducing power activity (163.1 ± 2.9 mg S₂O₃^2 − equivalent g^− 1 protein), which were directly related to disulfide bonds content of peptides (r = 0.9982 and 0.9216, respectively). pH was a good alternative to select bioactive peptides from wheat gluten hydrolysate.     

Three novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) using digestive proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from muscle of cuttlefish (Sepia officinalis) by treatment with various digestive proteases were investigated. The most active hydrolysate was obtained with the crude protease extract from the hepatopancreas of cuttlefish (64.47 ± 1.0% at 2 mg of dry weight/ml) with a degree of hydrolysis of 8%. By gel filtration on Sephadex G-25 and RP-HPLC on C18 column, three novel peptides with high ACE-inhibitory activity were purified and their molecular masses and amino acid sequences were determined. The three peptides Val-Tyr-Ala-Pro, Val-Ile-Ile-Phe and Met-Ala-Trp with IC₅₀ values of 6.1, 8.7 and 16.32 μM, respectively, were novel ACE-inhibitory peptides. Lineweaver–Burk plots suggest that the three purified peptides act as non-competitive inhibitors against ACE. These results suggest that some peptides from cuttlefish could be a beneficial ingredient for nutraceuticals against hypertension.     

Antihypertensive peptides from skimmed milk hydrolysate digested by cell-free extract of Lactobacillus helveticus JCM1004

In the present experiments, the proteinase, aminopeptidase and x-prolyl-dipeptidyl aminopeptidase activitives were measured at 513 ± 11.9, 376 ± 9.3 and 23.6 ± 1.6 units per gramme of cell-free extract of Lactobacillus helveticus JCM1004. ACE-inhibitory activity of skimmed milk hydrolysate produced by cell-free extract of L. helveticus JCM1004 was determined, and the optimum pH and hydrolysis time for the production of ACE-inhibition substances from skimmed milk were 6.5–7.0 and 6–10 h, respectively. Peptides of Val-Pro-Pro and Ile-Pro-Pro with potent ACE inhibitor and antihypertensive activitives were purified from the hydrolysate by three step-reverse-phase high-performance liquid chromatography. The IC₅₀ values of Val-Pro-Pro and Ile-Pro-Pro were 9.13 ± 0.21 and 5.15 ± 0.17 μM, respectively. Significant decrease (p < 0.01) in SBP of SHR was measured after a single gastric intubation of Val-Pro-Pro or Ile-Pro-Pro at 8 or 4 h, respectively.     

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC₅₀ value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.     

Isolation and identification of antimicrobial peptides derived by peptic cleavage of whey protein isolate

The antimicrobial potential of whey protein isolate hydrolyzed by gastrointestinal enzymes was determined by attempting to identify and characterize the antimicrobial peptides responsible. While tryptic and chymotryptic hydrolysates did not show antibacterial activity, whey proteins hydrolyzed for 45–90 min by pepsin exhibited significant activity. Fractionation of 60-min hydrolysate by reversed-phase high performance liquid chromatography yielded 5 fractions that were antibacterial, with minimum inhibitory concentrations comprised between 20 and 35 μg/mL. These fractions contained short peptides not previously identified as antimicrobial. Fragment 14–18 (KVAGT) of β-lactoglobulin is very close to a sequence previously identified as antibacterial and is found in antimicrobial sequences of diverse origin. Five other peptides derived from β-lactoglobulin, and one fragment from α-lactalbumin (f117–121, KVGIN), were also identified as antibacterial. The identified peptides do not match pepsin action exactly, indicating modified proteolysis of unknown origin. Protein by-products of the dairy industry offer potential for large-scale production of antimicrobial peptides.     

Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry

Loach protein was hydrolyzed by papain to obtain antioxidative peptides. The results showed that the loach protein hydrolysate (LPH) could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC₅₀ = 17.0 ± 0.54 mg/mL) and hydroxyl radicals (IC₅₀ = 2.64 ± 0.29 mg/mL). It could chelate cupric ion and inhibit the lipid peroxidation in a linoleic acid emulsion system. The hydrolysate was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to an electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude LPH. Therefore, it is possible to produce natural antioxidative peptides from loach protein by enzymatic hydrolysis and purification.