Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

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.     

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.     

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).     

Chemical composition,antioxidant activity and anti‐lipase activity of Origanum vulgare and Lippia turbinata essential oils

This study reported the chemical composition, phenolic content, antioxidant and anti‐lipase activity of oregano and Lippia essential oils. The major compounds found in oregano essential oil were γ‐terpinene (32.10%), α‐terpinene (15.10%), p‐cymene (8.00%) and thymol (8.00%). In Lippia essential oil, α‐limonene (76.80%) and 1,8‐cineole (4.95%) represented the major compounds. Oregano essential oil had higher phenolic content (12.47 mg gallic acid mL^?1) and DPPH scavenging activity (IC₅₀ 0.357 μg mL^?1) than Lippia essential oil (7.94 mg gallic acid mL^?1 and IC₅₀ 0.400 μg mL^?1, respectively). Both essential oils had similar antioxidant indexes (about 1.2) determined by Rancimat. Moreover, oregano essential oil had also higher anti‐lipase activity (IC₅₀ 5.09 and 7.26 μg mL^?1). Higher phenolic content in the essential oils was related with higher scavenging and anti‐lipase activities. Oregano and Lippia essential oils could be used as natural antioxidants on food products.     

Activities and sequences of the angiotensin I‐converting enzyme (ACE) inhibitory peptides obtained from the digested lentil (Lens culinaris) globulins

The aim of this study was the identification of potentially bioaccessible ACE‐inhibitory peptides obtained by in vitro gastrointestinal digestion of lentil globulins. ACE‐inhibitory peptides were purified by ion exchange chromatography and gel filtration. After the first step of purification, three peptide fractions with potential antihypertensive properties were obtained and the highest inhibitory activity was determined for the fraction 5 (IC₅₀ = 0.02 mg mL^?1). This fraction was separated on Sephadex G10, and six peptide fractions were obtained. The peptides of fraction (5‐F) with the highest potential antihypertensive activity (IC₅₀ = 0.13 mg mL^?1) were identified using ESI‐MS/MS. The sequences of peptides were KLRT, TLHGMV and VNRLM. Based on Lineweaver–Burk plots for the fraction 5‐F, the kinetic parameters as K_m (1.24 mm ), V_max (0.012 U min^?1), K_i (0.12 mg mL^?1) and mode of inhibition were determined.     

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.     

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.     

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.     

Phytochemicals content,antioxidant and hypoglycaemic activities of commercial nutmeg mace (Myristica fragrans L.) and pimento (Pimenta dioica (L.) Merr.)

Commercially dried powder of nutmeg mace (Myristica fragrans) and pimento (Pimenta dioica) spices was investigated for their high performance liquid chromatography phenolic profile and their antioxidant and hypoglycaemic properties by α‐amylase and α‐glucosidase inhibition tests. Generally, mace showed the most promising activity. An interesting protection of lipid peroxidation with an IC₅₀ value of 7.7 μg mL^?1 was found. A significant result was also obtained in ferric reducing ability power assay if compared to the positive control butylated hydroxytoluene (IC₅₀ value of 68.7 μg mL^?1 vs. 63.2 μg mL^?1, respectively). Mace also exhibited the highest carbohydrate‐hydrolysing enzyme inhibitory activity with IC₅₀ values of 62.1 and 75.7 μg mL^?1 against α‐amylase and α‐glucosidase, respectively. Overall, these results support the use of these spices not only as flavouring agent but also as food preservative and functional ingredients.     

Effects of physicochemical factors and in vitro gastrointestinal digestion on antioxidant activity of R‐phycoerythrin from red algae Bangia fusco‐purpurea

R‐phycoerythrin (R‐PE) was purified from the red algae Bangia fusco‐purpurea after 35–50% ammonium sulphate fractionation followed by ion‐exchange column chromatography on DEAE‐Sepharose, resulting in a purity (A₅₆₅/A₂₈₀) ratio of 5.1. The circular dichroism spectroscopy results suggested that the structure of R‐PE is predominately helical. The antioxidant activity of R‐PE was studied and revealed changes in conformation and antioxidant activity at different temperatures and pH values. After in vitro‐simulated gastrointestinal (GI) digestion of R‐PE, the scavenging activity of ABTS radical (EC₅₀, 769.9 μg mL^?1), DPPH radical (EC₅₀, 421.9 μg mL^?1), hydroxyl radical (EC₅₀, 32.4 μg mL^?1) and reducing power (A₇₀₀ = 0.5, 625.8 μg mL^?1) were measured. Gel filtration chromatography analysis showed that the molecular weight distribution of the final GI digest that still contained high antioxidant activity was <3 kDa. Our present results indicate that digestion‐resistant antioxidant peptides of R‐PE may be obtained by in vitro GI proteinases degradation.     

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.     

Inhibitory activity towards human α‐amylase in wheat flour and gluten

Wheat α‐amylase inhibitors (AI) have been targeted as potential triggers of noncoeliac gluten or wheat sensitivity (NCGS). The aim of this study was to determine AI activity towards α‐amylase from human source in wheat cultivars. Contrary to barley, buckwheat, or oats, high level of AI activity was found in wheat and, to a lesser extent, rye. AI activity (mean IC₅₀ = 137 μg mL^?1) did not vary with respect to ancient or recently developed wheat cultivars. Vital wheat gluten had very high and heat‐stable AI activity (mean IC₅₀ = 23 μg mL^?1), higher than wheat starch (?10 000 μg mL^?1) or acarbose (40 μg mL^?1), a medication for the management of hyperglycaemia and potentially causing digestive disorders due to the accumulation of undigested carbohydrates in the intestine. Data suggest that eating raw wheat gluten, flour or dough could pose a health risk.     

Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds

This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC₅₀ = 0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions.     

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.     

Production of whey protein isolate hydrolysate fractions with enriched ACE-inhibitory activity

A study on the enrichment of angiotensin-converting enzyme (ACE) inhibitory activity in whey protein isolate (WPI) hydrolysate fractions is presented. A previously identified low molecular mass fraction (1 kDa permeate) of an enzymatically hydrolysed heat-treated WPI with elevated ACE-inhibition (IC₅₀ = 0.23 g L⁻¹) was subjected to cascade membrane ultrafiltration (UF) and diafiltration steps at lab-scale. Assaying for ACE-inhibition revealed that the 1 kDa retentate demonstrated the highest ACE-inhibitory activity (IC₅₀ = 0.17 g L⁻¹). Isoelectric focussing (IEF) of the hydrolysate fraction further increased ACE-inhibition in fractions collected within the pH range 6.1–6.6. Overall, both UF and IEF enriched the ACE inhibitory activity in the original fraction by ∼52%, demonstrating the potential for enrichment of bio-functional activities in enzymatic hydrolysates of whey proteins.     

Antioxidant activity of predigested protein obtained from a range of farmed edible insects

This study investigated the antioxidant activities of peptides obtained by in vitro gastrointestinal digestion of edible insects. The antioxidant potential of edible insects' hydrolysates was determined as the free radical‐scavenging activity, ion chelating activity and reducing power. The highest antiradical activity against DPPH^˙, whose IC₅₀ value is the lowest, was noted for Amphiacusta annulipes (19.1 μg/mL) and that against ABTS^˙+ was the highest for Zophobas morio (4.6 μg/mL). The peptides obtained from A. annulipes also showed the highest Fe²⁺ chelation ability (58.82%) and reducing power (0.652). The highest ability to chelate Cu²⁺ was noted for Locusta migratoria (86.05%). The locust was characterised by the highest concentration of peptides before digestion and after digestion (3.13 and 5.88 mg/mL, respectively), and the DH was 36.29%.     

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.     

Comparative study of the essential oils of three Beilschmiedia species and their biological activities

This study was designed to examine the chemical compositions of the essential oils from three Beilschmiedia species and antioxidant, antimicrobial, antityrosinase, acetylcholinesterase and anti‐inflammatory activities. The essential oils of B. kunstleri, B. maingayi, B. penangiana gave β‐caryophyllene (10.6–12.1%), β‐eudesmol (17.5–24.1%) and δ‐cadinene (17.5–28.7%) as the most abundant components respectively. The bark oil of B. maingayi showed the highest activity in β‐carotene/linoleic acid (125.9%) and phenolic content (288.2 mg GA g^?1), while B. penangiana bark oil was found to have strong activity in DPPH (IC₅₀ 84.7 μg mL^?1) and ABTS (IC₅₀ 108.3 μg mL^?1). The essential oils of B. penangiana showed the best activity against Candida glabrata with MIC value 31.3 μg mL^?1. The bark oil of B. penangiana gave 82.5% tyrosinase inhibiton. The leaf oil of B. maingayi gave the highest inhibition in AChE (66.6%) and lipoxygenase (77.0%) assay. Our findings demonstrate that the essential oils have great potential for applications in pharmaceutical and food industries.     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.