Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product

To produce and identify bioactive peptides, two commercial enzymes, orientase (OR) and protease XXIII (PR) were used to hydrolyze tuna dark muscle by-product for up to 6 h, and the hydrolysates were evaluated for antioxidative properties. The results showed that, 60-min OR and 120-min PR hydrolysates possessed the highest antioxidative activity. Then, the protein hydrolysates were subjected to a Sephadex G-25 gel filtration chromatography, and the molecular weight of the peptide fractions which showed the highest antioxidative activity ranged from 390 to 1400 Da. The peptide fractions were further isolated using the two-step high-performance liquid chromatography (HPLC-1 and HPLC-2), and the amino acid sequences of the two antioxidative peptides from OR and PR hydrolysates were Leu–Pro–Thr–Ser–Glu–Ala–Ala–Lys–Tyr (978 Da) and Pro–Met–Asp–Tyr–Met–Val–Thr (756 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna dark muscle by-product may be useful ingredients in food and nutraceutical applications.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Functionalities and antioxidant properties of protein hydrolysates from the muscle of ornate threadfin bream treated with pepsin from skipjack tuna

Functional properties and antioxidant activities of protein hydrolysates prepared from ornate threadfin bream (Nemipterus hexodon) muscle, using skipjack tuna pepsin, with different degrees of hydrolysis (DH: 10%, 20% and 30%), were evaluated. Emulsifying and foaming properties of hydrolysates were governed by their DH and concentrations used. Hydrolysates with 20% DH had the highest scavenging activities for ABTS and DPPH radicals. However, chelating activity of hydrolysates for ferrous ion increased as DH increased. Size exclusion chromatography of the hydrolysate with 20% DH using Sephadex G-25 revealed that antioxidative peptides with molecular weight of approximately 1.3 kDa exhibited the highest ABTS radical-scavenging activity. In vitro simulated gastrointestinal digestion indicated that ABTS radical-scavenging activity of the antioxidative peptides was not affected by pepsin hydrolysis, whilst further digestion by pancreatin enhanced the activity. Therefore, protein hydrolysate from the muscle of ornate threadfin bream produced by skipjack tuna pepsin can be used as a promising source of functional peptides with antioxidant properties.     

Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides

Four peptides with high angiotensin-converting enzyme (ACE) inhibitory effect were separated from beef sarcoplasmic protein hydrolysates using commercial enzymes. They were identified as GFHI, DFHING, FHG, and GLSDGEWQ and their 50% inhibition concentration (IC₅₀) values against ACE were 117, 64.3, 52.9, and 50.5 μg/ml, respectively. These peptides were synthesised and further biological activities of these four peptides were measured, including antimicrobial, cytotoxic effect against cancer cells, and macrophage-stimulating effect. Peptide GLSDGEWQ showed growth inhibition on Salmonella Typhimurium, Bacillus cereus, Escherichia coli, and Listeria monocytogenes at a 100 ppm level but not on Staphylococcus aureus and Pseudomonas aeruginosa. Peptide GFHI showed higher inhibition activity on the growth of E. coli and P. aeruginosa at concentrations of 200 and 400 μg/ml. However, peptide FHG inhibited only P. aeruginosa at 200 and 400 μg/ml. The effect of separated peptides on breast cancer (MCF-7), lung cancer (A549), and stomach cancer (AGS) cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Peptide GFHI showed a slight decrease of MCF-7 cell viability in a dose dependent manner. When 400 μg/ml of peptide GFHI was applied to the AGS cell, its viability was decreased by 75%. However, peptide DFHINQ seemed to act as a nutrient to AGS cell because it increased its viability. None of the four peptides had a cytotoxic effect on A549 cells. Nitric oxide (NO) production of peptide GFHI by stimulation of macrophage was investigated at 100, 300, and 1000 μg/ml concentration. NO was not produced in all treatments. From these results it is expected that the ACE inhibitory peptides identified from beef sarcoplasmic protein hydrolysates have both antimicrobial and cancer cell cytotoxic effects.     

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.     

Characteristics of Bellamya purificata snail foot protein and enzymatic hydrolysates

The foot muscle protein of Bellamya purificata (mud snail, named Luosi in Chinese) was investigated. Its conformation change and increase in solubility were researched during enzymatic hydrolysis. The protein conformation was looser following an increase in pH (from 10 to 12), while the β-sheet was the main conformation at pH 12. Blending, ultrasonic extraction, ultradispersing and alkaline treatment increased the solubility of the foot muscle protein. The effects of several proteases on its hydrolysis were compared and Proleather FG-F was chosen. The relative molecular mass distribution, the free amino acids (FAA) content and the angiotensin-I converting enzyme (ACE) inhibitory activity of the hydrolysates were quantitatively analyzed and compared. In the Proleather FG-F hydrolysates, the percentage of the peptides with molecular weight between 150 and 2000 Da were 84.65%, much more than that in the Alcalase 2.4L hydrolysates (68.44%). Proleather FG-F released much less FAA (5.80%), than Alcalase 2.4L (17.01%). The IC₅₀ of the Proleather FG-F hydrolysate was 0.69 mg/ml, whereas for the Alcalase 2.4L hydrolysate the value was 3.30 mg/ml. Finally, response surface methodology (RSM) was used to optimize the factors (pH, enzyme: substrate ratio- E/S- and temperature) affecting Proleather FG-F hydrolysis.     

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC₅₀ = 287 μg/mL) and (ii) from β-lactoglobulin (IC₅₀ = 128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC₅₀).     

Antioxidant activity of hydrolysates obtained from scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle

Scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle were hydrolysed with commercially available food-grade proteases. The resulting hydrolysates showed DPPH and hydroxyl radicals scavenging abilities, reducing power, and ferrous ion chelating capacity. The antioxidant activities of hydrolysate of abalone foot muscle (HAFM) increased with increasing incubation time during the whole hydrolysis process in 180 min. Whereas, the antioxidant activities of hydrolysate of scallop adductor muscle (HSAM) increased at initial stage and peaked after 25-30 min of hydrolysis, and then gradually decreased thereafter. Compared with HAFM, HSAM with comparable hydrolysis time contained more free amino acids (FAA) and small-sized peptides (below 500 Da), which may account for the differences in antioxidant activities versus hydrolysis time curves of the two hydrolysates. The above results indicate that limited hydrolysis of proteins can increase their antioxidant activity, whereas extensive hydrolysis can decrease it.     

Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity

Gelatin obtained from giant squid (Dosidicus gigas) inner and outer tunics was hydrolyzed by seven commercial proteases (Protamex, Trypsin, Neutrase, Savinase, NS37005, Esperase and Alcalase) to produce bioactive hydrolysates. The Alcalase hydrolysate was the most potent angiotensin-converting enzyme (ACE) inhibitor (IC₅₀ = 0.34 mg/mL) while the Esperase hydrolysate showed the highest cytotoxic effect on cancer cells, with IC₅₀ values of 0.13 and 0.10 mg/mL for MCF-7 (human breast carcinoma) and U87 (glioma) cell lines, respectively. The radical scavenging capacity of gelatin increased approximately 3-fold for Protamex, Neutrase and NS37005 hydrolysates and between 7 and 10-fold for Trypsin, Savinase, Esperase and Alcalase hydrolysates. Trypsin, Savinase, Esperase and Alcalase hydrolysates had a metal chelating capacity above 80% whereas Protamex, Neutrase and NS37005 hydrolysates registered less than 25%. The antioxidant activity measured by FRAP (ferric ion reducing power) was largely unaffected by the enzyme used, increasing approximately 2-fold for all hydrolysates. The most active hydrolysates (Alcalase and Esperase) were comprised mostly of peptides with molecular weights ranging from 500 to 1400 Da, however, a clear relationship between bioactive properties and molecular weight distribution of all the hydrolysates was not fully established.     

Biological activity and chemical composition of different berry juices

Total phenolics, total anthocyanins, mineral content, radical scavenging activity and antiproliferative activity against human cancer cell lines were evaluated in fresh pressed juices of five different berries. Total phenolic content ranged from 133.0 to 260.3 mg of gallic acid equivalents/100 g of fresh weight, for red currant and black currant, respectively. Bilberry juice contained the highest amount of total anthocyanins (0.18%). Significant correlation between total phenolics content and radical scavenging activity was observed (r = −0.980; p < 0.01). All examined juices showed antiproliferative activity in dose-dependent manner with IC₅₀ ranging from 10.2 to 70.5 μl/ml. Black currant juice was the most effective inhibitor of proliferation in all cell lines tested (HeLa, Fem X, LS 174, MCF-7 and PC-3). Significant correlations of acidity and total anthocyanin content with antiproliferative activity of berry juices on HeLa cells, Fem X cells and MCF-7 cells were noticed. Berry juices are good sources of some minerals and contribute significantly to daily intake of these micronutrients.     

Transglutaminase cross-linking effect on sensory characteristics and antioxidant activities of Maillard reaction products from soybean protein hydrolysates

To improve the yield of Maillard peptides, a microbial transglutaminase (MTGase) was used to increase the content of 1000–5000 Da peptides in soybean protein hydrolysates by using a cross-linking reaction. The sensory characteristics and antioxidant activities of corresponding Maillard Reaction Products (MSPC) was then evaluated. After cross-linking treatment the content of 1000–5000 Da peptides in protein hydrolysates and the yield of Maillard peptides increased by 21.19% and 8.71%, respectively, which contributed to the improved mouthfulness of MSPC. The bitter amino acids were significantly decreased and the umami acids were markedly increased in MSPC. Volatile compounds identified by GC–MS analysis showed that the content of the important meaty flavour compounds (such as 2-methyl-3-furanthiol, bis(2-methyl-3-furyl)disulfide) of MSPC were dramatically higher than that of MRPs from uncross-linking peptides. Combined with sensory evalution, it was confirmed that MTGase cross-linking improved the flavour Characteristics and did not affect the antioxidant activity of MSPC.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Prediction of short peptides composition by RP-HPLC coupled to ESI mass spectrometry

The combination of reversed-phase chromatography and electrospray mass spectrometry was used to predict the amino acid composition of low molar weight peptides present in a complex mixture of rapeseed protein hydrolysates. First, amino acid retention times on a hydrophobic stationary phase were evaluated to determine hydrophobicity coefficients for each amino acid and then global hydrophobicity coefficients were established for standard peptides. In this way, a correlation between peptide hydrophobicity coefficients and retention time was established. Then, a C language program was developed to calculate all potential amino acid combinations from the mass of a peptide (molar mass < 1000 Da) and determine theoretical hydrophobicity coefficients corresponding to these combinations. Comparison between the theoretical retention time determined by the model and the experimental retention time resulted in the establishment of a sorted potential composition table. This methodology has been applied to two different rapeseed protein hydrolysates peptides that have been purified and sequenced.     

Temperature effect on the non-volatile compounds of Maillard reaction products derived from xylose–soybean peptide system: Further insights into thermal degradation and cross-linking

Solutions of soybean peptide with or without xylose were heated over a range of temperatures (80–130 °C) for 2 h to investigate the characteristics of Maillard reaction and the effect of temperature on amino acids and peptides. It was found that both peptide degradation and peptide cross-linking occurred in the Maillard reaction. The critical temperature for peptide degradation was 100 °C, and above it, peptides degraded quickly in thermal degradation system. However, in Maillard reaction system, peptides cross-linked rapidly when the temperature reached 110 °C. Bitter amino acids and peptides below 1000 Da decreased 18.44% and 28.49%, respectively, after Maillard reaction at 120 °C. On the other hand, peptides between 1000 and 5000 Da were increased significantly, nearly doubled compared to its initial hydrolysates after Maillard reaction at 120 °C. Moreover, the increase of macromolecule products was also accompanied with severe browning and pH decrement.     

Purification and identification of antihypertensive peptides from seaweed pipefish (Syngnathus schlegeli) muscle protein hydrolysate

Angiotensin-I-converting enzyme (ACE-I) inhibitory peptides were purified from the seaweed pipefish muscle protein using papain, alcalase, neutrase, pronase, pepsin and trypsin. Among them, the alcalase hydrolysate exhibited the highest ACE-I inhibitory activity. The alcalase hydrolysate was separated into four fractions (Fr1, Fr2, Fr3, and Fr4) by fast protein liquid chromatography (FPLC) on a Hiprep 16/10 DEAE FF anion exchange column. Among four fractions, Fr3 has shown the highest ACE-I inhibitory activity and it was further purified into three fractions (Fr3-I, Fr3-II, and Fr3-III) using reverse-phase high performance liquid chromatography (RP-HPLC) on a Primesphere 10 C18 (20 × 250 mm) column. The Fr3-II has exhibited the highest ACE-I inhibition (IC₅₀, 0.62 mg/ml) than the Fr3-III (IC₅₀, 1.44 mg/ml). The amino acid sequences of the obtained peptides from Fr3-II and Fr3-III were identified as Thr-Phe-Pro-His-Gly-Pro (MW, 744 Da) and His-Trp-Thr-Thr-Gln-Arg (MW, 917 Da) respectively. Furthermore, cell viability assay showed that no cytotoxicity of alcalase hydrolysate on human lung fibroblasts cell line (MRC-5). These results suggest that peptides derived from seaweed pipefish can be developed as antihypertensive ingredients in functional foods.     

Some functional properties of oat bran protein concentrate modified by trypsin

Oat bran protein concentrate (OBPC) was prepared from oat bran, and hydrolyzed using trypsin. Protein hydrolysates of three different degrees of hydrolysis (4.1%, 6.4% and 8.3% respectively) were obtained. SDS-PAGE analysis demonstrated that oat globulin was the major protein component in OBPC. After trypsin treatment, acidic polypeptides were partly degraded into large peptides (M_r = 29,000–33,000) and small peptides (M_r < 20,000); however, basic polypeptides were almost intact. The functional properties of the resulting products were compared with those of control OBPC. Marked changes in the protein functionality were caused by proteolysis. The solubility, water-holding capacity, emulsifying activity and foaming ability of the hydrolysates gradually increased with the increase in DH. However, the oil-holding capacity, emulsifying stability and foaming stability of the hydrolysates reduced to a certain extent.     

Enzymatic hydrolysis of heated whey: iron-binding ability of peptides and antigenic protein fractions

This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50°C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (β-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of β-lactoglobulin, α-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO₄·7H₂O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC.     

Angiotensin I-converting enzyme inhibitory properties of lentil protein hydrolysates: Determination of the kinetics of inhibition

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of two lentil varieties by in vitro gastrointestinal simulation, Alcalase/Flavourzyme, papain and bromelain. Protein/peptide profiles studied by electrophoresis and HPLC-SEC showed a rich composition of the hydrolysates in small peptides ranging in size from 0.244 to 1.06 kDa. ACE inhibitory activity was measured using the HPLC Hippuryl-His-Leu (HHL) substrate method. Significantly different (P < 0.05) IC₅₀ values ranging between 0.053 and 0.190 mg/ml were obtained for different hydrolysates. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (K_i) between 0.16 and 0.46 mg/ml. These results demonstrate that hydrolysates of lentil proteins obtained by different enzymatic digestions may contain bioactive components.     

Angiotensin I-converting enzyme inhibitory properties and SDS-PAGE of red lentil protein hydrolysates

Several research studies have shown that protein hydrolysates from milk and soy contain peptides that possess angiotensin I converting enzyme (ACE) inhibitory properties and may help to prevent hypertension. To date, no studies have been conducted to determine if red lentil (Lens culinaris) proteins contain peptides with ACE-inhibitory properties. The objective of the present work was to characterize the proteins present in red lentils and determine if tryptic hydrolysis could liberate peptides with ACE-inhibitory properties. Red lentil protein extracts were prepared and fractionated to obtain enriched albumin, legumin and vicilin fractions. Protein/peptide profiles were studied by electrophoresis and ACE-inhibitory activity was measured using the HPLC hippuryl-His-Leu (HHL) substrate method. Our results revealed that red lentil protein hydrolysates posses ACE-inhibitory properties. Furthermore, we demonstrated that the ACE-inhibitory property of the hydrolysates varied as a function of the protein fraction with the total lentil protein hydrolysate having the lowest half maximal inhibitory concentration (IC₅₀) (111 ± 1 μmol/L) (i.e., highest ACE-inhibitory activity), followed by the enriched legumin (119 ± 0.5 μmol/L), albumin (127 ± 2 μmol/L) and vicilin (135 ± 2 μmol/L) fractions, respectively.     

Hypocholesterolaemic effect of Bifidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate

Hypercholesterolaemia is one of the most important risk factors in the development of cardiovascular disease. In this study, we report the in vitro potential of Bifidobacterium animalis subsp. lactis (Bb12) cultures and bovine casein hydrolysates formed by trypsin and Bb12 culture to reduce cholesterols levels. Cholesterol levels in vitro were reduced by up to 48% after incubation with Bb12 and up to 87% after incubation with trypsin hydrolysates, whereas unhydrolysed bovine casein did not affect cholesterol levels. Individual peptide fractions, obtained from size-exclusion chromatography, from casein hydrolysates formed by trypsin after a 48 h hydrolysis, reduced cholesterol levels by 2.7–50%. The molecular masses of these fractions, containing hypocholesterolaemic peptides, were below 1200 Da, as determined by LC-MS.