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.     

Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

The chelating peptide (GPAGPHGPPG) derived from Alaska pollock skin enhances calcium,zinc and iron transport in Caco‐2 cells

Gly‐Pro‐Ala‐Gly‐Pro‐His‐Gly‐Pro‐Pro‐Gly (GPAGPHGPPG), a chelating peptide derived from Alaska pollock skin, has been approved with outstanding ability to chelate calcium, zinc and iron ions. In this study, the stability of GPAGPHGPPG during in vitro gastrointestinal enzymatic digestion, the potential binding site between peptide and metal ions as well as its effects on mineral transport in Caco‐2 cells were investigated. Results showed that approximately 75% of GPAGPHGPPG remained intact at the end of the in vitro gastrointestinal enzymatic digestion. The formation of peptide‐metal complex was potentially related to histidine and cyclic structure in terms of dehydration. Moreover, GPAGPHGPPG revealed significant promotional effects on calcium (112.7%, P < 0.01), zinc (32.3%, P < 0.01) and iron (27.7%, P < 0.01) transport in Caco‐2 cell monolayer. In conclusion, GPAGPHGPPG could be developed as a potential functional ingredient to prevent mineral deficiency.     

γ‐Glu‐Met synthesised using a bacterial glutaminase as a potential inhibitor of dipeptidyl peptidase IV

This study describes γ‐glutamyl dipeptides as competitive inhibitors of dipeptidyl peptidase‐IV (DPP‐IV), and the feasible synthesis of γ‐Glu‐Met through transpeptidation catalysed by a commercial glutaminase of Bacillus amyloliquefaciens. γ‐Glu‐Met, γ‐Glu‐Leu, γ‐Glu‐Phe, γ‐Glu‐Trp or γ‐Glu‐Tyr exhibited a competitive inhibitory effect on DPP‐IV. The yield of γ‐Glu‐Met was 26.16% under optimised conditions: 200 mm Gln, 20 mm Met, 0.1 U mL^?1 enzyme, pH 9.0, 37 °C and reaction time 3 h. For the first time, the side products containing characteristic sequences, that is poly‐γ‐glutamyl short chains with a terminal Met residue (γ‐Glu‐γ‐Glu‐Met and γ‐Glu‐γ‐Glu‐γ‐Glu‐Met) were identified. The superiority of the commercial glutaminase in the synthesis of DPP‐IV‐inhibitory peptides can enable the application of this novel process for manufacturing γ‐glutamyl‐peptides as potential functional ingredients in the type 2 diabetic diet.     

Antioxidant activity in HepG2 cells,immunomodulatory effects in RAW 264.7 cells and absorption characteristics in Caco‐2 cells of the peptide fraction isolated from Dendrobium aphyllum

DA‐P, fraction of peptides with a molecular weight <1 kDa isolated from Dendrobium aphyllum, was analysed in three types of cell lines to verify its bioactivity and absorptivity. The cellular antioxidant activity of DA‐P in HepG2 cells was used and results revealed an EC50 of 2.88 ± 0.143 mg mL^?1 and a CAA unit of 63.46 ± 2.11 μm QE/100 g peptides. DA‐P treatment enhanced the secretion of cytokines in RAW 264.7 cells. After demonstrating the presence of tight junctions in Caco‐2 monolayers, the absorption was 25.57% ± 0.016% and 19.7% ± 0.012% from different sides. The relatively high absorption indicated that the antioxidant‐relevant immune functions of DA‐P had a greater possibility to be absorbed by Caco‐2 cells. Free amino acids and LC‐MS/MS analysis indicated the degradation and expulsion of components after the absorption of DA‐P, and Ser‐Ser‐Arg was able to come across the monolayers.     

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

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

Determination of S‐methyl‐L‐methionine (SMM) from Brassicaceae Family Vegetables and Characterization of the Intestinal Transport of SMM by Caco‐2 Cells

The objectives of the current study were to determine S‐methyl‐L‐methionine (SMM) from various Brassicaceae family vegetables by using validated analytical method and to characterize the intestinal transport mechanism of SMM by the Caco‐2 cells. The SMM is well known to provide therapeutic activity in peptic ulcers. The amount of SMM from various Brassicaceae family vegetables ranged from 89.08 ± 1.68 μg/g to 535.98 ± 4.85 μg/g of dry weight by using validated ultra‐performance liquid chromatography‐electrospray ionization‐mass spectrometry method. For elucidating intestinal transport mechanism, the cells were incubated with or without transport inhibitors, energy source, or a metabolic inhibitor. Phloridzin and verapamil as inhibitors of sodium glucose transport protein (SGLT1) and P‐glycoprotein, respectively, were not responsible for cellular uptake of SMM. Glucose and sodium azide were not affected by the cellular accumulation of SMM. The efflux ratio of SMM was 0.26, implying that it is not effluxed through Caco‐2 cells. The apparent coefficient permeability (P _app) of SMM was 4.69 × 10^?5 cm/s, indicating that it will show good oral absorption in in vivo .     

Antioxidant Peptide Fractions Isolated from Wheat Germ Protein with Subcritical Water Extraction and Its Transport Across Caco‐2 Cells

Wheat germ protein (WGP) was extracted with subcritical water and then hydrolyzed with Alcalase 2.4 L to obtain antioxidant hydrolysates. Wheat germ peptides (WG‐P, Mw < 1 kDa) were purified by using Sephadex G‐15 column chromatography. The results showed that WG‐P‐4 possessed the strongest DPPH radical scavenging activity in comparison with other peptides fractions. In addition, free amino acids and LC‐MS/MS analysis showed that Gly‐Pro‐Phe, Gly‐Pro‐Glu, and Phe‐Gly‐Glu were the major peptides of WG‐P‐4. Interestingly, the WG‐P‐4 fractions had good absorption characteristic. Moreover, the ratio of P_app both sides of apical compartment (AP) and basolateral compartment (BL) were between 0.5 and 1.0 on Caco‐2 cell model, which indicated that transmembrane transportation was mainly passive transport. Therefore, WG‐P could exert an effective antioxidant action by across the intestinal epithelium.     

Studies on protein and its high‐molecular‐weight subunit composition in relation to chapati‐making quality of Indian wheat cultivars

In order to predict the suitability of wheat for chapati making, 15 Indian wheat cultivars were studied for various protein characteristics in relation to chapati‐making quality. The cultivars varied considerably in their protein characteristics and chapati‐making potential. Results clearly indicated that both quantitative and qualitative characteristics of proteins influenced the chapati‐making potential of cultivars. Puffed height, the important qualitative parameter of chapati, was positively correlated with protein content (r = 0.62, p < 0.05), gluten content (r = 0.79, p < 0.01), sodium dodecyl sulphate (SDS) sedimentation value (r = 0.57, p < 0.05) and Glu‐1 quality scores of high‐molecular‐weight (HMW) subunits (r = 0.66, p < 0.01). Overall quality score of chapati was positively correlated with gluten content (r = 0.64, p < 0.01), SDS sedimentation value (r = 0.60, p < 0.05) and Glu‐1 score (r = 0.58, p < 0.05). HMW subunit composition varied considerably among cultivars. Cultivars having 5 + 10 subunits at the Glu‐1D chromosome, a protein content of about 130 g kg^?1 and SDS sedimentation value around 75 ml yielded excellent chapatis, while those having 2 + 12 subunits, a protein content of about 115 g kg^?1 and SDS sedimentation value around 55 ml resulted in poor chapatis. Interestingly, the presence of the 1BL/1RS chromosome in cultivars had no adverse effect on chapati quality. © 2003 Society of Chemical Industry     

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.     

Bioactive components,ABTS radical scavenging capacity and physical stability of orange,yellow and purple sweet potato (Ipomoea batatas) powder processed by convection‐ or vacuum‐drying methods

This study aims to investigate the antioxidant properties and physical stability of convection‐ or vacuum‐dried orange, yellow and purple sweet potato (Ipomoea batatas) powder upon room temperature storage at different relative humidity (RH) of 33%, 43%, 54% and 75% for 25 days. Both convection and vacuum‐dried samples had lower total phenolic content (TPC), anthocyanin content and antioxidant activity compared to the fresh samples. Vacuum‐dried powder retained higher antioxidant activity than convection‐dried powder. Storage of the powder at high RH of 75% resulted in apparent clump formation, which was likely attributed to the decrease of glass transition temperature (T_g). In general, purple sweet potato powder contained the highest TPC (255.0 mg GAE 100 g DW^?1) and antioxidant activity (1924.0 μmol TEAC 100 g DW^?1), while orange powder had the highest beta‐carotene content (127.2 mg 100 g DW^?1). Vacuum‐dried sweet potato powder, which has relatively higher antioxidant activity, could be a potential functional ingredient or natural colourant for the food industry.     

Effect of in vitro‐simulated gastrointestinal digestion on the stability and antioxidant activity of blueberry polyphenols and their cellular antioxidant activity towards HepG2 cells

In this study we investigated the influence of in vitro‐simulated gastrointestinal digestion on the bioaccessibility and antioxidant activity of polyphenols from blueberries (Vaccinium spp.). Total phenolic, anthocyanin, and flavonoid content was determined, and extract and digesta compositions were analysed by HPLC‐ESI‐MS/MS. The phenolic compounds were relatively stable under a gastric environment, whereas polyphenols and anthocyanins were unstable under an intestinal environment. The bioaccessibility of polyphenol, anthocyanin, and flavonoid was greatly decreased after the intestinal digestion, and the recoveries were only 13.93%, 1.95%, and 15.68% (the IN sample), respectively. Polyphenolic profile alteration occurred during in vitro‐simulated gastrointestinal digestion. Changes of phenolic compound antioxidant activity during digestion correlated with polyphenol, flavonoid, and caffeic acid concentrations. Digested extract cellular antioxidant activity was lower than non‐digested extract activity (P < 0.05). Polyphenol dose–response correlations with cellular antioxidant activity were observed. These results indicated that in vitro‐simulated gastrointestinal digestion significantly impact polyphenols and their antioxidant activity.     

Effect of Acylglycerol Composition and Fatty Acyl Chain Length on Lipid Digestion in pH‐Stat Digestion Model and Simulated In Vitro Digestion Model

In this study, a pH‐stat digestion model and a simulated in vitro digestion model were employed to evaluate the digestion degree of lipids depending on different acylglycerols and acyl chain length (that is, diacylglycerol [DAG] compared with soybean oil representing long‐chain triacylglycerol compared with medium‐chain triacylglycerol [MCT]). In the pH‐stat digestion model, differences were observed among the digestion degrees of 3 oils using digestion rate (k), digestion half‐time (t_1/2), and digestion extent (Φmax). The results showed the digestion rate order was MCT > soybean oil > DAG. Accordingly, the order of digestion half‐times was MCT < soybean oil < DAG. In simulated in vitro digestion model, digestion rates (k′) and digestion half‐times (t′_1/2) were also obtained and the results showed a digestion rate order of MCT (k′ = 0.068 min^?1) > soybean oil (k′ = 0.037 min^?1) > DAG (k′ = 0.024 min^?1). Consequently, the order of digestion half‐times was MCT (t′_1/2 = 10.20 min) < soybean oil (t′_1/2 = 18.74 min) < DAG (t′_1/2 = 29.08 min). The parameters obtained using the 2 models showed MCT was digested faster than soybean oil, and that soybean oil was digested faster than DAG.     

Esterification of Quercetin Increases Its Transport Across Human Caco‐2 Cells

Plant polyphenols showed useful biochemical characteristics in vitro; however, the assessments of their clinical applications in vivo are restricted by their limited bioavailability due to their strong resistance to 1st‐pass effects during absorption. In order to improve the bioavailability of quercetin (QU), the ester derivative of QU (3,3′,4′,5,7‐pentahydroxy flavones, TAQU) was synthesized, followed by examining the oil–water partition coefficient as well as the transport mechanisms of QU and its ester derivative (TAQU) using human Caco‐2 cells. The transport characteristics of QU and TAQU transport under different conditions (different concentrations, time, pH, temperature, tight junctions, and potential transporters) were systematically investigated. Results showed that QU had a lower permeability coefficient (2.82 × 10^?6 cm/s) for apical‐to‐basolateral (AP‐BL) transport over 5 to 50 μM, whereas the transport rate for AP to BL flux of TAQU (5.23 × 10^?6 cm/s) was significantly greater than that of QU. Paracellular pathways were not involved during the transport of both QU and TAQU. QU was poorly absorbed by active transport, whereas TAQU was mostly absorbed by passive diffusion. Efflux transporters, P‐glycoproteins, multidrug resistance proteins were proven to participate in the transport process of QU, but not in that of TAQU. These results suggested that improving the lipophicity of QU by esterification could increase the transport of QU across Caco‐2 cells.     

Assessment of iron bioavailability from twenty elite late‐maturing tropical maize varieties using an in vitro digestion/Caco‐2 cell model

An in vitro digestion/Caco‐2 cell model was used to assess iron bioavailability of twenty elite late‐maturing tropical maize varieties grown in three diverse agroecologies in West and Central Africa (WCA). Kernel‐iron concentration of the varieties, averaged across locations, varied from 19.2 to 24.4 mg kg^?1, while mean kernel‐zinc concentration ranged between 19.4 and 24.6 mg kg^?1. Significant differences in iron bioavailability were observed among varieties, but the environment had no significant effect. Mean bioavailable iron ranged between 14% below and 43% above the reference control variety, TZB‐SR. Variety DMR‐LSR‐Y with the highest concentrations of kernel‐iron and ‐zinc of 24–25 mg kg^?1 across the three locations had a similar quantity of bioavailable iron as the reference control, TZB‐SR. In the long run this variety could be potentially effective in reducing iron deficiency because of its high kernel‐iron. The most promising varieties were Mid‐altitude STR synthetic and ACR91SUWAN‐1‐SRC1. They had kernel‐iron and ‐zinc levels of 22–24 mg kg^?1 and bioavailable iron 24–36% higher than the reference control, TZB‐SR. Additional research is necessary to determine if the increases in kernel‐iron concentration and bioavailable iron observed in this study can significantly improve the iron status of individuals in WCA at risk for iron deficiency. Copyright © 2004 Society of Chemical Industry     

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.     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Antioxidant potential of protein‐rich serum from farmed swan goose (Anser cygnoides): in vitro and in vivo evaluation of antioxidant effects

As food ingredient, Anser cygnoides is farmed in large scale; however, its blood is underused. The characteristics, stability and antioxidant activities of P owder of farmed C ygnoides S erum (FACSP) were investigated. Results showed that FACSP was protein‐rich and displayed satisfactory antioxidant activities. In vitro analysis indicated that the IC₅₀ values of 2, 2′‐azinobis‐(3‐ethylbenz‐thiazoline‐6‐sulphonate), 1,1‐diphenyl‐2‐picrylhydrazyl and hydroxyl radicals were 1.56, 2.76 and 39.55 mg mL^?1, respectively. In vivo experiment showed that the activity of total superoxide dismutase and content of glutathione of the FACSP‐treated groups were enhanced, and the contents of malondialdehyde and protein carbonyl were decreased. The parameters of 400 and 800 mg kg^?1 bw day^?1 dose groups were equally or approximately to vitamin C. The FACSP shows potential as an antioxidant functional food at a dose of 400 mg kg^?1 bw day^?1.     

Antioxidant,immunomodulatory and antiproliferative effects of gelatin hydrolysates from seabass (Lates calcarifer) skins

This study investigated the antioxidant, immunomodulatory and antiproliferative potentials of gelatin hydrolysates from seabass skins in cell model systems. Gelatin hydrolysates were extracted from seabass skins using different processes and enzyme concentrations. The ability of the hydrolysates to protect against H₂O₂‐induced DNA damage was assessed on U937 cells using the Comet assay, and one of the samples showed DNA protective effects. All samples showed immunomodulatory potential by significantly (P < 0.05) reducing interleukin‐6 (IL‐6) and IL‐1β production in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. Antiproliferative activities of seabass skin hydrolysates were measured using human colon cancer (Caco‐2) and liver cancer (HepG2) cell lines as the model cell cultures. The inhibition of cell proliferation of Caco‐2 and HepG2 cancer cells occurred in a dose‐dependent manner at concentrations of 1–25 mg mL^?1. Therefore, seabass skin hydrolysates prepared using an appropriate process could serve as a potential functional food ingredient with various health benefits.     

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.