Antioxidant activity of ovine casein hydrolysates: identification of active peptides by HPLC–MS/MS

This paper shows the potential role of different ovine casein fractions and their hydrolysates to exert antioxidant activity. ABTS^·+ decolorization assay was used to evaluate the antioxidant activity of the casein fractions (β-, κ- and α_s-caseins) before and after their hydrolysis by pepsin, trypsin and chymotrypsin. Although the antioxidant activity increased in all the fractions after hydrolysis, the effect was particularly remarkable in the κ-casein fraction, which increased its antioxidant activity almost threefold. Further assays in a linoleic acid oxidation system showed that κ-casein hydrolysate inhibited lipid peroxidation. Analysis of the ovine κ-casein hydrolysate by RP-HPLC–MS/MS allowed the identification of 12 peptide sequences with potential antioxidant properties. One of the most abundant peptides, the fragment HPHPHLSF [f(98–105)] was chemically synthesized. Results showed that this κ-casein-derived peptide was a potent inhibitor of linoleic acid oxidation with an activity similar to that obtained with the synthetic antioxidant BHT. Although other peptides might also contribute, HPHPHLSF was the one most likely to be responsible for the activity found in the κ-casein hydrolysate.     

Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.     

Preparation and characterisation of an easily absorbabl Mg‐casein hydrolysate complex produced through enzymatic hydrolysis and ultrafiltration

In this study, we synthesised a Mg‐casein hydrolysate complex that allowed the effective absorption of Mg. The type of enzyme (papain, alcalase 2.4 L, pepsin, trypsin) and the enzyme/substrate ratio for casein hydrolysis was optimised. When the enzyme/substrate ratio was 30%, the alcalase 2.4 L‐hydrolysate showed the highest Mg‐chelation efficiency, of 96.1%. To characterise and enhance the function of casein hydrolysate, we fractionated the casein hydrolysate according to molecular weight using ultrafiltration. The Mg‐chelation efficiency was increased with the decrease in the molecular‐weight range of the hydrolysate fractions. The smallest casein hydrolysate (fraction 5, 1 kDa<) is used for preparation of Mg‐casein hydrolysate complex. Synthesised Mg‐casein hydrolysate complex (fraction 5) exhibited 100% Mg solubility and 39.5% Mg bioavailability. These results indicated that the Mg‐casein hydrolysate remained a stable chelate during simulated gastro‐intestinal digestion in vitro. The Mg‐casein hydrolysate complex exhibited excellent antioxidant activity as well as Mg binding.     

Positively charged peptides from casein hydrolysate show strong inhibitory effects on LDL oxidation and cellular lipid accumulation in Raw264.7 cells

Inhibiting low-density lipoprotein (LDL) oxidation and cellular lipid accumulation to reduce foam cell formation plays a key role in preventing atherosclerosis. Casein hydrolysate (66% < 1 kDa) was separated into four different charged fractions. The inhibitory effect of peptide fractions on LDL oxidation and cellular lipid accumulation was analysed using a CuSO₄ cell-free system and a Cu²⁺-mediated and ox-LDL-induced Raw264.7 macrophage cell-based system. Casein peptide fractions not only significantly inhibited LDL oxidation but also prevented cellular lipid accumulation. Positively charged fractions exhibited stronger inhibitory effects than negatively charged fractions. Seven peptides with different charge properties were synthesised. With the increase in net positive charge, the ability of peptides to inhibit LDL oxidation was enhanced. Peptides containing lysine presented better inhibition than those that contained histidine. This study suggests that casein hydrolysate, especially positively charged peptide fractions, could be used as a natural antioxidant in functional foods to prevent atherosclerosis.     

Identification and molecular mechanism of action of antibacterial peptides from Flavourzyme-hydrolyzed yak casein against Staphylococcus aureus

Antibacterial peptides can be released from yak milk casein. To date, the amino acid sequences and mechanism of action of yak casein–derived antibacterial peptides remain unknown. The current study identified antibacterial peptides from yak casein and their molecular mechanism of action. Our results showed that yak α-casein, β-casein, and κ-casein could be effectively hydrolyzed by Flavourzyme (Solarbio Science and Technology Co. Ltd.), and the 2-h hydrolysate showed the highest antibacterial rate of 43.07 ± 2.59% against Staphylococcus aureus. The 1,000 to 3,000 Da fraction accounted for 23.61% of the 2-h hydrolysate and had an antibacterial rate of 62.64 ± 4.40%. Three novel peptides with antibacterial activity were identified from this fraction, and the β-casein–derived peptide APKHKEMPFPKYP showed the strongest antibacterial effect (half-maximal inhibitory concentration = 0.397 mg/mL). Molecular docking predicted that APKHKEMPFPKYP interacted with 2 important enzymes of Staph. aureus, dihydrofolate reductase and DNA gyrase, through hydrophobic, hydrogen bonding, salt bridge, and π-π stacking interactions. Our findings suggest that the yak casein–derived peptides may serve as a potential source of natural preservatives to inhibit Staph. aureus.     

Iron-chelating activity of chickpea protein hydrolysate peptides

Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption.     

Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus)

Mackerel (Scomber austriasicus) hydrolysates were prepared by an autolytic process and accelerated hydrolysis with a commercial enzyme, Protease N. Changes in the levels and compositions of free amino acids and small peptides during hydrolysis were investigated to find out their relationships with antioxidant activities. Increased levels of free amino acids, anserine, carnosine and other peptides of the hydrolysates obtained with protease were much higher than those by autolysis. Different antioxidant measurements including the inhibition of linoleic acid autoxidation, the scavenging effect on ,-diphenyl-β-picrylhydrazyl free radical, and the reducing power showed that mackerel hydrolysates possessed noticeable antioxidant activities. A good correlation existed between the amount of peptides and antioxidant activity. Three peptide fractions were separated from the hydrolysate by size exclusion chromatography. Results revealed that the peptide with molecular weight of approximately 1400 Da possessed a stronger in vitro antioxidant activity than that of the 900 and 200 Da peptides.     

Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization–mass spectrometry

The porcine skin collagen was hydrolyzed by different protease treatments to obtain antioxidative peptides. The hydrolysate of collagen by cocktail mixture of protease bovine pancreas, protease Streptomyces and protease Bacillus spp. exhibited the highest antioxidant activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, metal chelating and in a linoleic acid peroxidation system induced by Fe²⁺. And degree of hydrolysis highly affected the antioxidant properties of the hydrolysates. Four different peptides showing strong antioxidant activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography and high-performance liquid chromatography. The molecular masses and amino acid sequences of the purified antioxidant peptides were determined using electrospray ionization (ESI) mass spectrometry. One of the antioxidative peptides, Gln-Gly-Ala-Arg, was then synthesized and the antioxidant activities measured using the aforementioned methods. The results confirmed the antioxidant activity of this peptide, and adds further support to its feasibility as a provider of natural antioxidants from porcine skin collagen protein.     

Identification of radical scavenging peptides (<3 kDa) from Burgos-type cheese

The aim of the present study was to identify low molecular weight peptides with radical scavenging activity from cheese made using different types of rennet: batch 1, animal rennet (95% chymosin and 5% bovine pepsin), batch 2, rennet of plant origin (Cynara cardunculus) and batch 3, microbial rennet (Mucor miehei). After preparation of the peptide extracts (<3 kDa), antioxidant activity was assayed by their DPPH radical scavenging and metal chelating a6ctivity. All of the batches showed antioxidant activity, which could be dependent on the peptides which are present in extracts: Batch 2 and 3 showed the highest values for DPPH inhibition and chelating effect. Fourteen fractions out of the total peptide fractions collected after RP-HPLC analysis showed radical scavenging activity using the DPPH inhibition method. Free amino acids and peptides were identified from these fractions. One of the peptides, derived from α_s1-casein, was a potential new antioxidant peptide. These antioxidant peptides were present in a lower content in extracts obtained from animal rennet cheese in comparison with the other extracts.     

Obtaining of Brassica carinata protein hydrolysates enriched in bioactive peptides using immobilized digestive proteases

Brassica carinata protein hydrolysates were obtained by sequential hydrolysis with immobilized trypsin, chymotrypsin, and carboxypeptidase A on glioxyl-agarose supports. The final protein hydrolysate with a 36% degree of hydrolysis was made up of peptides smaller than 15 kDa. Three peptide fractions were obtained after gel filtration chromatography and antioxidative, hypocholesterolemic, and inhibitory of angiotensin converting enzyme activities were assayed in comparison with the starting materials (protein isolate and hydrolysate). Total protein hydrolysate achieved the best results in the reduction of micellar cholesterol and fraction II, composed by peptides between 1800 and 1400 Da, showed the best antioxidant and inhibitory of angiotensin converting enzyme activities. These results show that B. carinata seed proteins may represent an useful source of bioactive peptides after hydrolysis with digestive proteases such as trypsin, chymotrypsin, and carboxypeptidase A.     

Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin

Sunflower protein hydrolysates obtained with pepsin and pancreatin were used for purification of copper-chelating peptides by affinity chromatography with copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of β-carotene oxidation in the presence of copper. The protein hydrolysate obtained after 180 min incubation with pepsin plus 60 min with pancreatin was the most inhibitory of β-carotene oxidation. Purified chelating peptides were 2.5 times more antioxidant than the parent protein hydrolysate. Chelating peptides were enriched in certain amino acids, such as histidine and arginine, with respect to the original hydrolysate. This work shows that chelating peptides may be generated during digestion of sunflower proteins and have a protective role, due to their antioxidative activity, and favour mineral bioavailability.     

Multifunctional peptides from egg white lysozyme

Hen’s egg white lysozyme (HEWL) is one of the major egg white proteins with well demonstrated antimicrobial activity. Bioactive peptides other than antimicrobial peptides from HEWL have not been reported; therefore, the purpose of the study was to explore new bioactivities of lysozyme-derived bioactive peptides. HEWL was hydrolysed with Alcalase and fractionated by cation-exchange chromatography. The Alcalase HEWL hydrolysate and its fractions were analyzed for inhibitory activities against calmodulin-dependent phosphodiesterase (CaMPDE) and antioxidant activities using oxygen radical absorbance capacity-fluorescein (ORAC-FL), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS⁺) and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) radical scavenging methods. The fractionated peptides had higher CaMPDE inhibition activity, ORAC-FL value and ABTS⁺ scavenging activity than those of the hydrolysate. Peptide sequences in the most overall active fractions were characterized by LC–MS/MS. Our results showed that HEWL hydrolysate and its peptide fractions may serve as useful ingredients in the formulation of functional foods and nutraceuticals.     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

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.     

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties

BACKGROUND: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin‐like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene‐related peptide (CGRP)‐like peptides demonstrated an increase in CGRP‐like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin‐converting enzyme (ACE)‐1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION: Fractionation of an FPH using membrane separation, with a molecular weight cut‐off adapted to the peptide composition, may provide an effective means to concentrate CGRP‐like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut‐off of the membrane only, as is currently done in the literature. Copyright © 2010 Society of Chemical Industry     

芝麻抗氧化肽分离纯化与结构鉴定

通过纳滤、超滤、制备液相对胰蛋白酶水解芝麻蛋白的酶解液中抗氧化肽进行分离纯化,随后采用高效液相质谱联用法进行结构鉴定,并合成相应多肽验证抗氧化活性。结果表明,经分离纯化与结构鉴定,共获得5个芝麻抗氧化肽,Glu-Leu-Phe-Phe-Gly-Ala-Gly-Gly- Glu-Asn-Pro-Glu-Ser-Phe-Phe-Lys(ELFFGAGGENPESFFK)、Phe-Glu-Ser-Glu-Ala-Gly-Leu- Thr-Glu-Phe-Trp-Asp-Arg(FESEAGLTEFWDR)、Asp-Val-Ala-Asn-Glu-Ala-Asn-Gln-Leu-Asp- Leu-Lys(DVANEANQLDLK)、Glu-Asn-IIe-Glu-His-Thr-Ala-Ala-Thr-His-Ser-Tyr -Asn-Pro- Arg(ENIEHTAATHSYNPR)、Gln-Asp-Asn-Ala-Asn-Asn-Ala-Asn-Gln-Leu-Asp-Pro-Asn-Pro- Arg(QDNANNANQLDPNPR)。胰蛋白酶酶解产生的芝麻抗氧化肽N端以酸性氨基酸残基为主,C末端为碱性氨基酸残基;除抗氧化肽ELFFGAGGENPESFFK是芝麻7S酶解产物以外,其余多肽均来自芝麻11S蛋白酶解。     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.     

The (193–209) 17‐residues peptide of bovine β‐casein is transported through Caco‐2 monolayer

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β‐casein (β‐CN, 193–209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β‐CN (193–209) peptide across Caco‐2 cell monolayer. In addition, we evaluated the possible routes of the β‐CN (193–209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β‐CN (193–209) peptide resisted the action of brush‐border membrane peptidases, and that it was transported through the Caco‐2 cell monolayer. The main route involved in transepithelial transport of the β‐CN (193–209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight‐junctions could not be excluded. Our results demonstrated the transport of an intact long‐chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.     

Antioxidant Activities of Roselle (Hibiscus Sabdariffa L.) Seed Protein Hydrolysate and its Derived Peptide Fractions

In this study, peptide fractions with strong antioxidant activity were obtained from Roselle seed protein hydrolysates. Pepsin followed by pancreatin were used to digest Roselle protein at different time in order to produce hydrolysate with antioxidant activity suitable for conversion to high-value products. The hydrolysates obtained after different hydrolysis times were analyzed for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The 3 h hydrolysate showed the highest antioxidant activity therefore, it was separated into four fractions (I, II, III, and IV) by gel filtration on Sephadex G-15. The antioxidant efficacies of the 3 h Roselle seed protein hydrolysates and its fractions were investigated using different in vitro methods. All fractions were effective antioxidants, with fraction III showing the strongest antioxidant activity. The fractions were then analyzed for amino acid composition. The analysis revealed that fraction III contained higher amounts of Serine, Glycine, Arginine, Alanine, Tyrosine, Valine, Phenylalanine, and Proline compared to the other fractions. Most of these amino acids have been reported to show antioxidant activity. The results showed that the hydrolysate derived from Roselle seed protein, particularly fraction III, could be a natural antioxidant source suitable for use as a food additive.