Pilot-scale production of hydrolysates with altered bio-functionalities based on thermally-denatured whey protein isolate

Whey protein isolate (WPI) solutions (100 g L⁻¹ protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase^® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC₅₀) in solution. The lowest ACE inhibition concentration (IC₅₀ = 0.23 g L⁻¹ protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.     

Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions

Casein and whey protein fractions from goat milk were hydrolysed by subtilisin and trypsin, individually and in combination, to release angiotensin converting enzyme (ACE)-inhibitory peptides. Selected hydrolysates were fractionated by size exclusion chromatography (SEC) and further characterised. The highest ACE-inhibitory activity was obtained from the casein fraction hydrolysed by the combination of enzymes. SEC presented 4 fractions with fraction F2 (<2.3 kDa) containing the highest concentration of peptides and the highest activity. F2 contained a number of peptides not previously identified from caprine caseins but with structural similarity to other ACE-inhibitory peptides. The most active fraction in relation to protein content was F4 with IC₅₀ between 9.3 and 5.1 μg mL⁻¹. This fraction contained a compound tentatively identified as WY, an active dipeptide not previously reported from caseins. The high inhibitory capacity of these fractions points towards the advantage of implementing a membrane process to concentrate the most active peptides.     

Enhancement of angiotensin I converting enzyme inhibitory activity and improvement of the emulsifying and foaming properties of corn gluten hydrolysate using ultrafiltration membranes

In this study, we attempted to enhance angiotensin I converting enzyme (ACE) inhibitory activity using an ultrafiltration (UF) membrane and to improve the emulsifying and foaming properties of fractions. ACE inhibitory activities of the corn gluten hydrolysate prepared by Flavourzyme had the same trend as protein contents and the IC₅₀ value was 0.18 mg solid after 8 h hydrolysis. The hydrolysate was separated by using two kinds of UF membrane (10 and 5 kDa cut-off membranes) and three fractions, >10 kDa, 5–10 kDa, and <5 kDa, were obtained. The yields of these three fractions were 58.3%, 27.2% and 14.5% as dry matter, respectively. The IC₅₀ value (the concentration of ACE inhibitor required to inhibit 50% of the ACE activity under specific conditions) of the <5 kDa fraction was 0.05 mg solid and was approximately fourfold that of the original, 5–10, and >10 kDa fractions. The average hydrophobicity of the <5 kDa fraction was slightly lower than that of the others according to the hydrophobicity index of Nozaki and Tanford, but according to the Krigbaum and Meirovitch index, that of the <5 kDa fraction was slightly higher than those of the others, showing there might be no significant differences among the fractions. The emulsifying activity and emulsion stability in the <5 kDa fraction was higher than those in the other fractions. Two fractions, <5 kDa and >10 kDa, showed poor foaming capacity at below pH 6.0. The foam stabilities of two fractions (<5 kDa and >10 kDa) increased with increasing pH, but that of 5–10 kDa decreased with increasing pH. The UF treatment was an effective method for the enhancement of ACE inhibitory activity with improvement of functional properties.     

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.     

Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from Thai jasmine rice bran protein hydrolysates

Rice bran protein hydrolysate (<50 kDa RBPH) from Thai jasmine variety demonstrating a high Angiotensin I converting enzyme (ACE) inhibitory activity was purified and characterised. ACE inhibitory peptides were obtained from a two-step purification process: gel filtration and preparative reverse-phase high-performance chromatography (RP-HPLC) and then identified by mass spectrometer hybrid quadrupole-time-of-flight. A novel peptide GSGYF in the RBPH was firstly identified and found to have a partial sequence homology of Oryza sativa Japonica Group. This sequence was further synthesised to exhibit as good an inhibition potency with IC₅₀ value of 2.11 µg mL⁻¹ as Captopril (1.15 µg mL⁻¹). The cytotoxicity test revealed that this RBPH is non-toxic against Vero cells. In addition, the <50 kDa RBPH was resistant to in vitro digestion by pepsin and trypsin. These findings suggest that the RBPH containing ACE inhibitory peptides is likely to be safer and healthier than synthetic drugs and can be an effective food supplement for lowering blood pressure.     

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.     

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).     

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.     

Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests

In vitro gastrointestinal digestion of pea and whey protein produced high angiotensin I converting enzyme (ACE) inhibitory activity with IC₅₀ values of 0.070 and 0.041 mg protein ml^?1 respectively. Ultrafiltration/centrifugation using a membrane with a molecular weight cut‐off of 3000 Da decreased the IC₅₀ value to 0.055 mg protein ml^?1 for pea permeate and 0.014 mg protein ml^?1 for whey permeate. Further fractionation by reverse phase HPLC gave IC₅₀ values as low as 0.016 mg protein ml^?1 for pea and 0.003 mg protein ml^?1 for whey. Consequently, these purification steps enriched the ACE inhibitory activity of the pea digest more than four times and that of the whey digest more than 13 times. HPLC profiles after digestion and ultrafiltration indicate that high ACE inhibitory activity is due to short and more hydrophobic peptides. The results also suggest that potent ACE inhibitory peptides were present alongside low active peptides in whey hydrolysate, while all peptides had more or less the same ACE inhibitory activity in pea hydrolysate. In addition, the hydrolysates and enriched fractions will resist in vivo gastrointestinal digestion after oral administration. Hence these ACE inhibitory peptides, as part of functional foods, can play significant roles in the prevention and treatment of hypertension. Copyright © 2004 Society of Chemical Industry     

Performance of whey protein hydrolysate–maltodextrin conjugates as emulsifiers in model infant formula emulsions

Model infant formula emulsions containing 15.5, 35.0 and 70.0 g L⁻¹ protein, soybean oil and maltodextrin (MD), respectively, were prepared. Emulsions were stabilised by whey protein hydrolysate (WPH) + CITREM (9 g L⁻¹), WPH + lecithin (9 g L⁻¹) or WPH conjugated with MD (WPH–MD). All emulsions had mono-modal oil droplet size distributions post-homogenisation with mean oil droplet diameters (D_4,3) of <1.0 μm. No changes in the D_4,3 were observed after heat treatment (95 °C, 15 min) of the emulsions. Accelerated storage (40 °C, 10 d) of unheated emulsions resulted in an increase in D_4,3 for CITREM (2.86 μm) and lecithin (5.36 μm) containing emulsions. Heated emulsions displayed better stability to accelerated storage with no increase in D_4,3 for CITREM and an increase in D_4,3 for lecithin (2.71 μm) containing emulsions. No increase in D_4,3 over storage was observed for unheated or heated WPH–MD emulsion, indicating its superior stability.     

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.     

Novel β-casein derived antioxidant and ACE-inhibitory active peptide from camel milk fermented by Leuconostoc lactis PTCC1899: Identification and molecular docking

Antioxidant and angiotensin-I converting enzyme inhibitory (ACE-I) activities of bioactive peptide fractions from camel milk fermented by Leuconostoc lactis PTCC 1899 were assessed. The fraction <3 kDa obtained from ultrafiltration showed ACE-I (IC₅₀ = 1.61 ± 0.18 mg mL⁻¹) and ABTS radical scavenging (1883.39 μm TE mg⁻¹ protein) activities and was purified through RP-HPLC. The active peptide, MVPYPQR, with antioxidant (8933.05 μm TE mg⁻¹ peptide) and ACE-I (IC₅₀ = 30 μm) activities was identified. To investigate the ACE-I mechanism of the purified peptide the docking study was performed. The presence of hydrogen bond between Gln 162 (S'1 pocket of ACE) and Arg in the C terminal of the peptide was identified and the peptide could distort the Zn²⁺ tetrahedral geometry of the enzyme. This study showed the ability of Leuc. lactis to produce a novel and safe functional food by hydrolysing the milk proteins during the fermentation.     

Inhibition of the in vitro activities of α-amylase, α-glucosidase and pancreatic lipase by yellow field pea (Pisum sativum L.) protein hydrolysates

The aim of this work was to produce yellow field pea protein-derived peptides as inhibitors of α-amylase, α-glucosidase and pancreatic lipase activities. A pea protein concentrate was hydrolysed with alcalase, chymotrypsin, pepsin or trypsin and the hydrolysates separated into different fractions (<1, 1–3, 3–5, 5–10 kDa) by membrane ultrafiltration. Peptide sequence analysis showed that the alcalase hydrolysate had higher levels of di- and tripeptides when compared with the chymotrypsin, pepsin and trypsin hydrolysates. The peptide fractions inhibited α-amylase and α-glucosidase activities at levels that were similar to the unfractionated hydrolysates. The peptides were more active against α-amylase (inhibition at μg level) than α-glucosidase (mg level). In contrast, the fractionated peptides had reduced ability (IC₅₀ >4.2 mg mL⁻¹) when compared with the unfractionated hydrolysate (IC₅₀ <4.2 mg mL⁻¹) to inhibit lipase activity. Enzyme kinetic studies revealed that the peptides reduced α-amylase activity through competitive inhibition. However, inhibition of α-glucosidase activity was non-competitive.     

Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract

The hydrolysis of bovine whey protein concentrate (WPC), α-lactalbumin (α-La) and caseinomacropeptide (CMP), by aqueous extracts of Cynara cardunculus, was optimized using response surface methodology. Degree of hydrolysis (DH), angiotensin-converting enzyme (ACE)-inhibitory activity and antioxidant activity were used as objective functions, and hydrolysis time and enzyme/substrate ratio as manipulated parameters. The model was statistically appropriate to describe ACE-inhibitory activity of hydrolysates from WPC and α-La, but not from CMP. Maximum DH was 18% and 9%, for WPC and α-La, respectively. 50% ACE-inhibition was produced by 105.4 (total fraction) and 25.6 μg mL⁻¹ (<3 kDa fraction) for WPC, and 47.6 (total fraction) and 22.5 μg mL⁻¹ (<3 kDa fraction) for α-La. Major peptides of fractions exhibiting ACE-inhibition were sequenced. The antioxidant activities of WPC and α-La were 0.96 ± 0.08 and 1.12 ± 0.13 μmol trolox equivalent per mg hydrolysed protein, respectively.     

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).     

Evaluation of various Bacillus coagulans isolates for the production of high purity L-lactic acid using defatted rice bran hydrolysates

Defatted rice bran (DRB) constitutes an abundant by-product stream, generated during rice milling and subsequent bran oil extraction. Enzymatic hydrolysis of starch and protein content in DRB was optimised in terms of solid loading. Among the four solid loadings evaluated (10%, 15%, 20% and 25%), the hydrolysate derived from 20% solids resulted in the highest concentration of glucose (82.3 g L⁻¹) and free amino nitrogen (234.8 mg L⁻¹). The fermentability of the hydrolysate was evaluated via screening of sixteen isolates. All the strains were able to grow and produce high purity L-lactic acid, utilising the DRB as sole carbon and nutrient source. Among the studied strains, the Bacillus coagulans A107 isolate presented the most promising results in terms of final lactic acid concentration (75.9 g L⁻¹), yield (0.90 g g⁻¹) and productivity (2.7 g L⁻¹ h⁻¹). The results of this study indicate that DRB could be employed as an inexpensive, alternative substrate for L-lactic acid production.     

Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions

Enzymatic hydrolysates from flaxseed protein were investigated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. Pepsin, ficin, trypsin, papain, thermolysin, pancreatin and Alcalase were used to hydrolyze flaxseed proteins followed by fractionation using ultrafiltration to isolate low-molecular-weight peptides, and separation of the Alcalase hydrolysate into cationic peptide fractions. Using N-(3-[2-furyl]acryloyl)-phenylalanylglycylglycine as substrate, the protein hydrolysates showed a concentration-dependent ACE inhibition (IC₅₀, 0.0275–0.151 mg/ml) with thermolysin hydrolysate and Alcalase cationic peptide fraction I (FI) showing the most potent activity. Flaxseed peptide fractions also showed no or moderate inhibitory activities against human recombinant renin (IC₅₀, 1.22–2.81 mg/ml). Kinetics studies showed that the thermolysin hydrolysate and FI exhibited mixed-type pattern of ACE inhibition whereas cationic peptide fraction II inhibited renin in uncompetitive fashion. These results show that the protein components of flaxseed meal possess peptide amino acid sequences that can be exploited as potential food sources of anti-hypertensive agents.     

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 °C and hydrolyzed using the enzymes Alcalase, α-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme α-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 °C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate.     

Polyphenolic composition,antioxidant and antiproliferative effects of wild and cultivated blackberries (Rubus fruticosus L.) pomace

The content of total polyphenolics, antioxidative capacity and antiproliferative activity were tested in wild and cultivated blackberry pomace. Wild blackberry pomace extract Tw2 showed the highest following contents: total polyphenolics (50.16 mg GAE g⁻¹ dw), flavonoids (7.73 mg Qc g⁻¹ dw), flavonols (6.63 mg Qc g⁻¹ dw) and total monomeric anthocyanins (13.40 mg Cy g⁻¹). Tw2 extract significantly inhibited free radicals: IC₅₀^DPPH = 127.76 μg mL⁻¹, IC₅₀^ABTS = 26.53 μg mL⁻¹ and IC₅₀ ^{˙ OH} = 168.62 μg mL⁻¹, and the growth of breast adenocarcinoma IC₅₀^MCF7 = 306.68 μg mL⁻¹ and cervix epitheloid carcinoma cell lines IC₅₀^HeLa = 315.49 μg mL⁻¹. Wild blackberry varieties had higher extraction yields, higher total polyphenolic contents and possessed stronger biological effects compared to cultivated blackberries (P < 0.05). All blackberry extracts showed high biological potential that could be attributed to high total polyphenols and flavonoids content and could be utilised as value-added functional food.     

Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems

Enzymatic hydrolysis was used for preparing hydrolysates from wheat gluten which is by-product during production of wheat starch. The enzyme used for the hydrolysis was papain. The hydrolysate was separated based on the molecular weight of the peptides by membrane ultrafiltration (UF) with a molecular weight cut-off of 5 kDa into permeate (P) and retentate (5-K) fractions. The antioxidative activities of the hydrolysate and its UF fractions were investigated by using the TBA method and scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The three fractions showed strong antioxidative activities in the linoleic acid oxidation system, and exhibited DPPH radical scavenging activity. The antioxidative activity of the P fraction was almost the same as that of vitamin E at pH 7.0. The molecular weight distribution of the P fraction was concentrated in 4.2 kDa (86.5%) after gel permeation chromatography fractionation using an HPLC system.The P and 5-K fractions had higher surface hydrophobicities (H₀) at pH7.0 compared with the hydrolysate. The resulting UF fractions were superior to the hydrolysate in terms of antioxidative activities.