Angiotensin I‐converting enzyme inhibitory peptides FQPSF and LKYPI identified in Bacillus subtilis A26 hydrolysate of thornback ray muscle

Angiotensin I‐converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC₅₀ of 0.83 mg mL^?1. To identify peptides responsible for this activity, the extract was eluted through size‐exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC₅₀ of 0.42 and 0.51 mg mL^?1, respectively. These fractions were analysed by nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC₅₀ of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides.     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.     

Isolation of peptides from a novel brewers spent grain protein isolate with potential to modulate glycaemic response

The in vitro dipeptidyl peptidase‐IV (DPP‐IV) inhibitory activity of a Brewers’ spent grain protein‐enriched isolate (BSG‐PI) Alcalase? hydrolysate (AlcH), which had previously been identified as a relatively potent angiotensin‐converting enzyme (ACE) inhibitor, was determined. The half maximal DPP‐IV inhibitory concentration (IC₅₀) value of AlcH following 240‐min digestion was 3.57 ± 0.19 mg mL^?1. Ultrafiltration fractionation did not significantly increase the DPP‐IV inhibitory activity of the AlcH fractions. Subjection of AlcH to simulated gastrointestinal digestion (SGID), which yielded SAlcH, resulted in a significant increase in DPP‐IV inhibitory activity (P < 0.05), particularly after the intestinal phase of digestion. Following semi‐preparative reverse phase high performance liquid chromatography (RP‐HPLC) fractionation of SAlcH, fraction 28 was identified as having highest mean DPP‐IV inhibitory activity. Two novel DPP‐IV inhibitory peptides, ILDL and ILLPGAQDGL, with IC₅₀ values of 1121.1 and 145.5 μm , respectively, were identified within fraction 28 of SAlcH following ultra‐performance liquid chromatography (UPLC)‐tandem mass spectrometry (MS/MS). BSG protein‐derived peptides were confirmed as having dual ACE and DPP‐IV inhibitory activities.     

Identification of angiotensin converting enzyme inhibitory and antioxidant peptides in a whey protein concentrate hydrolysate produced at semi‐pilot scale

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were identified in a 5 kDa ultrafiltration permeate of a whey protein hydrolysate generated at semi‐pilot scale. Further laboratory scale ultrafiltration of this 5 kDa permeate resulted in a 0.65 kDa permeate with antioxidant, (1.11 ± 0.074 μmol TE per mg dry weight, oxygen radical absorbance capacity, ORAC) and ACE inhibitory (ACE IC₅₀ 0.215 ± 0.043 mg mL^?1) activities. Semi‐preparative (SP) reverse phase high‐performance liquid chromatography (RP‐HPLC) of the 0.65 kDa permeate resulted in a fraction (SP_F3) with a 4.4‐fold increase in ORAC activity (4.83 ± 0.45 μmol TE mg dry weight) and a 1.3‐fold increase in ACE inhibitory activity (84.35 ± 1.36% inhibition when assayed at 0.28 mg mL^?1). Peptides within SP_F3 were identified using UPLC‐ESI‐MS/MS. Met‐Pro‐Ile had the highest ORAC activity (205.75 ± 12.08 μmol TE per mmol peptide) while Met‐Ala‐Ala and Val‐Ala‐Gly‐Thr had the highest ACE inhibitory activities (IC₅₀:515.50 ± 1.11 and 610.30 ± 2.41 μm , respectively).     

Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates

Microwave pretreatment and hydrolysis were applied to rainbow trout (Oncorhynchus mykiss) by-products to produce bioactive peptides with dual in vitro angiotensin-I converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV) inhibitory activities. Peptides were fractionated using the single step electrodialysis with ultrafiltration membrane (EDUF). Concentration of cationic peptides (CP) increased in the recovery solution, reaching 125 μg mL⁻¹ after a 4-h treatment with migration rate of 15.68 ± 2.98 g m⁻² h. CP fractions displayed ACE and DPP-IV I inhibitory properties, with IC₅₀ values of 0.0036 mg mL⁻¹ and 1.23 mg mL⁻¹ respectively. The bioactivity was attributed to the low molecular weight peptides (300–500 Da) recovered. CP exhibited non-competitive inhibition patterns for ACE and DPP-IV, which were dose dependent. These results showed that bioactive peptides can successfully be separated from complex hydrolysate mixtures by EDUF. The fractionated peptides can serve as potential functional food ingredients or nutraceuticals for the management of hypertension and diabetes.     

Effects of fermentation conditions on the potential anti‐hypertensive peptides released from yogurt fermented by Lactobacillus helveticus and Flavourzyme®

This study investigates the effects of fermentation conditions on the production of angiotensin‐converting enzyme inhibitory (ACE‐I) peptides in yogurt by Lactobacillus helveticus 881315 (L. helveticus) in the presence or absence of Flavourzyme^®, which is derived from a mould, Aspergillus oryzae and used for protein hydrolysis in various industrial applications. Optimal conditions for peptides with the highest ACE‐I activity were 4% (v/w) inoculum size for 8 h without Flavourzyme^® supplementation, and 1% inoculum size for 12 h when combined with Flavourzyme^®. The yogurt fermented by L. helveticus resulted in IC₅₀ values (concentration of inhibitor required to inhibit 50% of ACE activity under the assayed conditions) of 1.47 ± 0.04 and 16.91 ± 0.25 mg mL^?1 with and without Flavourzyme^® respectively. Seven fractions of ACE‐I peptides from the yogurt incorporated with L. helveticus and Flavourzyme^® were separated using the preparative high‐performance liquid chromatography. Fraction (F3) showed the highest ACE‐I activity with an IC₅₀ of 35.75 ± 5.48 μg mL^?1. This study indicates that yogurt may be a valuable source of ACE‐I peptides, which may explain the outcomes observed in the experimental and clinical studies and foresee the application of fermented milk proteins into functional foods or dietary supplements.     

Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity,antioxidant activities and physicochemical profiles

In this study, grass carp peptides were prepared by enzymatic hydrolysis of grass carp protein using the combination of Alcalase and Neutrase, and angiotensin‐I converting enzyme (ACE) inhibitory activity in vitro, antihypertensive activity in vivo, antioxidant activities, and physicochemical properties of peptides achieved from grass carp protein were characterised after ultrafiltration and desalted processes using mixed ion exchange resins. The purified peptides exhibited strong ACE inhibitory activity (IC₅₀ = 105 μg mL^?1), antihypertensive activity with the maximal drop for systolic blood pressure (SBP) of 43 mmHg at a dosage of 100 mg per kg body weight in spontaneously hypertensive rat (SHR), and antioxidant activities indicated by thiobarbituric acid‐reactive substance values in a liposome‐oxidising system, radical‐scavenging activity and chelation of metal ions (Fe²⁺). The molecular weight of peptides was <1000 Da. Compared to grass carp protein, the peptides separated from enzymatic hydrolysates possessed similar amino acid compositions, but contained higher concentrations of essential amino acids. Moreover, the peptides exhibited excellent solubility at a wide range of pH values from 2 to 10, and lower apparent viscosity than the protein. The peptides separated from enzymatic hydrolysates might be used as a promising ingredient in antihypertensive functional foods and nutraceuticals.     

Bioactive properties of enzymatic hydrolysates from abdominal skin gelatin of yellowfin tuna (Thunnus albacares)

Gelatin (90.6 ± 0.1%) was optimally prepared by response surface methodology from yellowfin tuna (Thunnus albacares, YT) abdominal skin. To investigate bioactive properties of enzymatic hydrolysates from the abdominal skin gelatin (ASG), ASG was hydrolysed with alcalase, protamex, neutrase and flavourzyme as affected by hydrolysis time. Antioxidant, nitrite scavenging and angiotensin‐I converting enzyme (ACE) inhibitory activities of the hydrolysates were determined. Antioxidant activities of the hydrolysates were found through linoleic acid peroxidation inhibitory effects. Alcalase‐derived hydrolysates (AHs) were more effective than others in metal ions chelating, superoxide anion scavenging and hydroxyl radical scavenging activities (P < 0.05). AHs showed significantly stronger nitrite scavenging activities (44.4–60.7%) than others (P < 0.05). Fraction A from AH showed strong ACE inhibitory activity (IC₅₀ of 0.75 mg mL^?1). These results suggest that YT ASG and its enzymatic hydrolysates could be functional food and/or pharmaceutical ingredients with potent antioxidant, anticarcinogenic and antihypertensive benefits.     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

Sweet potato protein hydrolysates: antioxidant activity and protective effects on oxidative DNA damage

In this study, sweet potato protein (SPP) hydrolysates were prepared by six enzymes (alcalase, proleather FG‐F, AS1.398, neutrase, papain and pepsin). The antioxidant activities and protective effect against oxidative DNA damage of SPP hydrolysates were investigated. Alcalase hydrolysates exhibited the highest hydroxyl radical‐scavenging activity (IC₅₀ 1.74 mg mL^?1) and Fe²⁺‐chelating ability (IC₅₀ 1.54 mg mL^?1) (P < 0.05). Compared with other five hydrolysates, the hydrolysates obtained by alcalase had the most abundant <3‐kDa fractions. In addition, below 3‐kDa fractions of alcalase hydrolysates showed the highest antioxidant activities and protective effects against DNA damage through both scavenging hydroxyl radicals and chelating Fe²⁺, which was probably because of the increase in several antioxidant amino acids, such as His, Met, Cys, Tyr and Phe, as well as the hydrophobic amino acids. The results suggested that enzymatic hydrolysis could be used as an effective technique to produce high value‐added peptides products from SPP.     

Purification and characterisation of antioxidant and nitric oxide inhibitory peptides from Tilapia (Oreochromis niloticus) protein hydrolysate

Nitric oxide (NO)‐inhibitory and antioxidative activities of tilapia hydrolysates were prepared using ultrasound pretreatment at 70 W for 30 and 45 min, respectively, followed by Flavourzyme hydrolysis for 1 h. Both hydrolysates were fractionated using size exclusion chromatography on Sephadex G‐25 column and purified by RP‐HPLC. The amino acid sequence of the most potent and purified fractions was determined using LC/MS/MS. The antioxidant peptide (KAFAVIDQDKSGFIEEDELKLFLQNFSAGARAGDSDGDGKIGVDEFAALVK, MW: 6334.49 KDa) and NO‐inhibitory peptide (AFAVIDQDKSGFIEEDELKLFLQNFSAGARAGDSDGDGKIGVDEFAALVK, MW: 6309.49 Da) produced no cytotoxicity in RAW264.7 macrophage cell lines at the concentration of 100 mg mL^?1. The purified peptides at the concentration 100 μg mL^?1 possessed antioxidative and NO‐inhibitory activities 83.0 ± 1.1% and 40.9 ± 0.2%, respectively, which were about 100 times those of their counterpart crude hydrolysates.     

Determination of reaction kinetics of hydrolysis of tilapia (Oreochromis niloticus) protein for manipulating production of bioactive peptides with antioxidant activity,angiotensin‐I‐converting enzyme inhibitory activity and Ca‐binding properties

To manipulate enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein for production of bioactive peptides, its reaction kinetics was intensively studied. The study showed that the production of peptides with different bioactive properties including antioxidant activity, angiotensin‐I‐converting enzyme (ACE) inhibition and Ca‐binding property and their kinetics were affected by the degree of hydrolysis and substrate concentration. A comparative study on reaction kinetics found that the kinetic parameters for the production of each bioactive peptide are unique, that is, the maximum initial velocity, V_max, for hydrolysis of protein was as high as 1.07 mg mL^?1 min^?1, but that for the production of peptides with antioxidant activity and Ca‐binding property were very low, range of 7.14–66.7 μg mL^?1 min^?1, and that for the production of peptides with ACE inhibitory activity was the lowest, at 2.57 μg mL^?1 min^?1. This knowledge of reaction kinetics of protein hydrolysis would be useful for manipulating and optimising the production of peptides with desired bioactive properties.     

The impact of fermentation and in vitro digestion on formation angiotensin converting enzyme (ACE) inhibitory peptides from pea proteins

Pea seeds were fermented by Lactobacillus plantarum 299v in monoculture under different time and temperature conditions and the fermented products were digested in vitro under gastrointestinal conditions. After fermentation and digestion ACE inhibitory activity was determined. In all samples after fermentation no ACE inhibitory activity was noted. Potentially antihypertensive peptides were released during in vitro digestion. The highest DH (68.62%) were noted for control sample, although the lowest IC₅₀ value (0.19 mg/ml) was determined for product after 7 days fermentation at 22 °C. The hydrolysate characterised by the highest ACE inhibitory activity was separated on Sephadex G10 and two peptides fractions were obtained. The highest ACE inhibitory activity (IC₅₀ = 64.04 μg/ml) for the first fraction was noted. This fraction was separated by HPLC and identified by LC–MS/MS and the sequence of peptide derived from pea proteins was determined as KEDDEEEEQGEEE.     

Antioxidant and anti‐acetylcholinesterase activities of anchovy (Coilia mystus) protein hydrolysates and their memory‐improving effects on scopolamine‐induced amnesia mice

Anchovy protein hydrolysates (APHs) were prepared through hydrolysis for 2, 4 or 8 h (APH‐2, APH‐4 and APH‐8, respectively). The chemical analyses, in vitro assessments [antioxidant activity and acetylcholinesterase (AchE) inhibitory activity] and in vivo mice tests were evaluated. Results revealed that APH‐8 exhibited the strongest reducing power and AchE inhibitory capacity (IC₅₀ = 159.76 ± 0.03 mg mL^?1), which may be due to its specific amino acid composition and newly formed peptides. In addition, AchE inhibitory kinetics of amino acids suggested that lysine was featured of both competitive and noncompetitive inhibitors. Furthermore, the results of in vivo study showed that all APHs exhibited memory‐improving action on scopolamine‐induced amnesia mice especially, APH‐8, indicating that anchovy protein is a potential source for health‐promoting peptides.     

Tannin fraction from Ampelopsis grossedentata leaves tea (Tengcha) as an antioxidant and α‐glucosidase inhibitory nutraceutical

Leaf of Ampelopsis grossedentata is a new resource of functional foods with healthful properties. Antioxidant and α‐glucosidase inhibitory activities of water extract (made in the style of drinking), tannin fraction (TF) and dihydromyricetin (DMY) from A. grossedentata leaves were evaluated. The main component of TF was identified as gallotannins. DPPH and ABTS radical scavenging activities and reducing power of TF were superior to those of water extract, however, inferior to those of DMY. In no PBS wash protocol of cellular antioxidant activity assay, DMY and TF exhibited similarly, while in PBS wash protocol, the value of TF was higher than that of DMY. In addition, TF possessed the highest α‐glucosidase inhibitory activities (IC₅₀ = 1.94 μg mL^?1), followed by water extract (IC₅₀ = 23.10 μg mL^?1) and DMY (IC₅₀ = 72.21 μg mL^?1). The strong α‐glucosidase inhibitory activity of TF may attribute to the binding capacity to enzymes, as confirmed by fluorescence analysis.     

Antioxidant,enzyme inhibitory and antiproliferative activity of polyphenolic‐rich fraction of commercial dry ginger powder

Polyphenolic‐rich fraction obtained from locally produced dry ginger powder in Brahmaputra valley, India, and commercially available dry ginger (Zingiber officinale) rhizome powder consisted of [6]‐gingerol (41.9%), [6]‐shogaol (24.3%), 1‐dehydro‐6‐gingerdione (8.6%), [8]‐gingerol (7.2%), [10]‐gingerol (5.1%), [6]‐paradol (5.9%) and [4]‐gingerol (3.6%). Traces of methyl‐[6]‐gingerol and methyl‐[8]‐gingerol (both at 1.8%) were also detected. The fraction exhibited high antioxidant capacity [total phenolics (TP), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA assay)], effectively inhibited isolated digestive enzymes (α‐glucosidase, pancreatic lipase and angiotensin converting enzyme) and inhibited the proliferation of colon (HT29; IC₅₀ of 1.06 ± 0.02 mg mL^?1) and gastric (AGS IC₅₀ of 1.29 ± 0.03 mg mL^?1) adenocarcinoma cells, without affecting the proliferation of their nontransformed counterparts (IC₅₀ > 2.0 mg mL^?1). This case study demonstrates that locally produced and commercially available dry ginger powder from Brahmaputra valley, India, retains numerous food components that may enhance human health.     

Angiotensin-converting enzyme inhibitory activity of hydrolysates generated from whey protein fortified with salal fruits (Galtheria shallon) by enzymatic treatment with Pronase from Streptomyces griseus

Whey proteins mixed with salal fruits extract (0–20% w/w) were hydrolysed with Pronase E from Streptomyces griseus for a period of 8 h. The angiotensin-converting enzyme (ACE) inhibitory activity of the hydrolysates was highest (IC₅₀: 0.087 mg mL⁻¹) for samples fortified with the highest extract concentration (20%). Peptides (>7 amino acids) with documented ACE inhibitory activity (DAQSAPLRVY, ALPMHIR, DKVGINY, LHLPLPL, YPFPGPI, YPFPGPIPN, VYPFPGPIPN) were identified bv LC-MS/MS data analysis using a database search approach. Fluorescence spectra of the whey proteins mixed with salal fruits extract indicates fluorescence quenching for α-lactalbumin. SDS-PAGE analysis suggests that α-lactalbumin is less susceptible to proteolysis when the extract is included in the formula. Data indicate that α-lactalbumin may be interacting with phenolic compounds naturally present in salal fruits. These interactions and the formation of complexes between a-Lac and phenolic compounds may affect the hydrolysis pattern of whey protein and the release of peptides with ACE-inhibitory activity.     

Preparation and identification of angiotensin I-converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure

Sweet potato protein hydrolysates (SPPH) with angiotensin I-converting enzyme (ACE) inhibitory activity were prepared by papain, pepsin and alcalase under high hydrostatic pressure (HHP, 100–300 MPa). HHP significantly increased degree of hydrolysis (DH), nitrogen recovery (NR) and molecular weight (MW) <3 kDa fractions contents of SPPH by all three enzymes (P < 0.05). MW < 3 kDa peptide fractions from SPPH by alcalase under 100 MPa showed the highest ACE inhibitory activity (IC₅₀ value 32.24 µg mL⁻¹), and was subjected to purification and identification by semi-preparative RP-HPLC and LC-MS/MS. Fifty-four peptides ranged from 501.28 to 1958.88 Da with 5–18 amino acids were identified and matched sporamin A and B sequences. Five identified peptides with sequences of VSAIW, AIWGA, FVIKP, VVMPSTF and FHDPMLR displayed good ACE inhibitory activity with the contribution of Val, Trp, Phe and Arg. Thus, SPPH by enzymatic hydrolysis under HHP can be potentially used in functional food.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

The influence of heat treatment of chickpea seeds on antioxidant and fibroblast growth‐stimulating activity of peptide fractions obtained from proteins digested under simulated gastrointestinal conditions

This study presents the effect of heat treatment of chickpea seeds on biological activity of peptides obtained by in vitro gastrointestinal digestion. The most significant antiradical activity against ABTS^+? expressed as IC₅₀ value was observed for 3.5‐ to 7‐kDa peptide fraction from TC hydrolysate (41.01 μg mL^?1). In turn, peptide fraction of 3.5–7.0 kDa obtained from raw chickpea seeds hydrolysate showed the highest antiradical activity against DPPH^? and Fe²⁺ chelating activity with IC₅₀ value of 20.94 and 52.53 μg mL^?1, respectively. The highest Cu²⁺ chelating activity was observed for peptides obtained from TC hydrolysate (IC₅₀ = 56.60 μg mL^?1). Peptide fraction <3.5 kDa from TC hydrolysate demonstrated the most significant reducing power (0.362 A₇₀₀/μg mL^?1). The peptide fraction of 3.5–7 kDa from TC hydrolysate also showed the highest fibroblast growth‐stimulating activity. These results indicated that the heat treatment process has no significant effect on antiradical activity against DPPH^? and Fe²⁺ chelating ability of peptides.