Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of the angiotensin I-converting enzyme-inhibitory peptides in Pacific hake (Merluccius productus) fillet hydrolysate

Fish protein hydrolysate (FPH) produced by incubation of Pacific hake fillet with 3.00% Protamex at pH 6.5 and 40 degrees C for 125 min demonstrated in vitro ACE-inhibitory activity (IC50 = 165 microg/mL), which was enhanced by ultrafiltration through a 10 kDa molecular weight cutoff membrane (IC50 = 44 microg/mL). However, after simulated gastrointestinal digestion, FPH and ultrafiltrate had similar ACE-inhibitory activity (IC 50 = 90 microg/mL), indicating that FPH peptides act as "pro-drug type" inhibitors and that enrichment by ultrafiltration may be unnecessary. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed that the molecular weights of major peaks were <1 kDa regardless of ultrafiltration. ACE-inhibitory activities of digested hydrolysates were not significantly affected by preincubation with ACE ( P > 0.05) and exhibited a competitive inhibitory mode. A permeability assay using fully differentiated colorectal adenocarcinoma (Caco-2) cells showed an apical to basolateral transport of peptides that ranged from approximately 2 to 20% after 2 h at 37 degrees C. Pacific hake fillet hydrolysates are a potentially bioavailable source of ACE-inhibitory peptides awaiting further in vivo study.     

Optimizing angiotensin I-converting enzyme inhibitory activity of Pacific hake (Merluccius productus) fillet hydrolysate using response surface methodology and ultrafiltration

The in vitro angiotensin I-converting enyzme (ACE) inhibitory activity of Pacific hake hydrolysates was investigated as a function of hydrolysis conditions, starting material variability, and ultrafiltration. Hake fillets were hydrolyzed using Protamex protease under various conditions of pH, hydrolysis time, and enzyme-to-substrate ratio (% E/S) according to a response surface methodology (RSM) central composite design. The hydrolysate produced at pH 6.5, 125 min, and 3.0% E/S had an IC 50 of 165 +/- 9 microg of total solids/mL. ACE-inhibitory activity was not significantly different (P < 0.05) for hydrolysates produced using higher time-enzyme combinations within the model or from fish of different catches. Ultrafiltration (10 kDa molecular mass cutoff) resulted in an IC50 value of 44 +/- 7 microg of peptides/mL, 2.5 times more potent than the commercial product PeptACE Peptides (IC50 = 114 +/- 8 microg of peptides/mL). These results suggest that hydrolysates prepared with minimal fractionation from Pacific hake, an undervalued fish, may be a commercially competitive source of ACE-inhibitory peptides.     

Production of cod (Gadus morhua) muscle hydrolysates. Influence of combinations of commercial enzyme preparations on hydrolysate peptide size range

The complement of enzyme activities of a selection of commercial protease preparations were determined using fluorogenic substrates. Alcalase was used in combination with other commercial enzyme preparations to produce cod muscle (Gadus morhua) hydrolysates. Each muscle hydrolysate was characterized with respect to the percentage degree of hydrolysis (DH %), peptide molecular weight range, and free amino acid content. The enzyme preparations containing predominantly protease or endopeptidase activities achieved high DH % and produced significant amounts of peptides below a molecular weight of 3000. Alcalase combined with exopeptidase-rich preparations produced hydrolysates rich in low-molecular-weight peptides. Selecting combinations of enzyme preparations with complementary activity profiles could be used to manipulate the peptide molecular weight profile of hydrolysates.     

Adsorption of whey protein isolate at the oil-water interface as a function of processing conditions: a rheokinetic study

In this paper we present surface dynamic properties (interfacial tension and surface dilational properties) of a whey protein isolate with a high content of beta-lactoglobulin (WPI) adsorbed on the oil-water interface as a function of adsorption time. The experiments were performed at constant temperature (20 degrees C), pH (5), and ionic strength (0.05 M). The surface rheological parameters and the interfacial tension were measured as a function of WPI concentration (ranging from 1 x 10(-)(1) to 1 x 10(-)(5)% w/w) and different processing factors (effect of convection and heat treatment). We found that the interfacial pressure, pi, and surface dilational modulus, E, increase and the phase angle, phi, decreases with time, theta, which should be associated with WPI adsorption. These phenomena have been related to diffusion of the protein toward the interface (at short adsorption time) and to the protein unfolding and/or protein-protein interactions (at long-term adsorption) as a function of protein concentration in solution and processing conditions.     

Structural changes imposed on whey proteins by UV irradiation in a continuous UV light reactor

The objective of this study was to investigate the structural changes of whey proteins during exposure in a continuous-flow UV reactor. Varying UV irradiation dosages were obtained by controlling the flow rate and the mixing speed. Whey protein isolate (WPI) solutions at concentrations of 1% and 5% (w/v) were circulated at flow rates ranging from 30 to 800 mL·min(-1), and changes in physicochemical properties of the proteins were investigated. Intrinsic fluorescence spectra and surface hydrophobicity measurements suggested changes in the tertiary structure of the proteins with UV exposure. The UV treatment also increased the concentration of total and accessible thiol groups in 1% WPI solutions, while no change was measured in 5% WPI solutions. Size-exclusion chromatography demonstrated the formation of UV-induced aggregates and oxidation products (N-formylkynurenine and dityrosine) of aromatic amino acids. Furthermore, the UV-induced changes in protein conformation increased the susceptibility of whey proteins to pepsin hydrolysis.     

Identification and characterization of topoisomerase II inhibitory peptides from soy protein hydrolysates

Topoisomerases are targets of several anticancer agents because their inhibition impedes the processes of cell proliferation and differentiation in carcinogenesis. With very limited information available on the inhibitory activities of peptides derived from dietary proteins, the objectives of this study were to employ co-immunoprecipitation to identify inhibitory peptides in soy protein hydrolysates in a single step and to investigate their molecular interactions with topoisomerase II. For this, soy protein isolates were subjected to simulated gastrointestinal digestion with pepsin and pancreatin, and the human topoisomerase II inhibitory peptides were co-immunoprecipitated and identified on a CapLC- Micromass Q-TOF Ultima API system. The inhibitory activity of these peptides from soy isolates toward topoisomerase II was confirmed using three synthetic peptides, FEITPEKNPQ, IETWNPNNKP,and VFDGEL, which have IC 50 values of 2.4, 4.0, and 7.9 mM, respectively. The molecular interactions of these peptides evaluated by molecular docking revealed interaction energies with the topoisomerase II C-terminal domain (CTD) (-186 to -398 kcal/mol) that were smaller than for the ATPase domain (-169 to -357 kcal/mol) and that correlated well with our experimental IC 50 values ( R (2) = 0.99). In conclusion, three peptides released from in vitro gastrointestinal enzyme digestion of soy proteins inhibited human topoisomerase II activity through binding to the active site of the CTD domain.     

Production, characterization, and application of anti-gossypol polyclonal antibodies

This paper describes the development of antibodies for detection of gossypol in cottonseed. Gossypol was linked to LPH (Limulus polyphemus hemolymph), BSA (bovine serum albumin), amines, or amino acids via Schiff base intermediates for the production of immunogen, solid-phase, and gossypol derivatives. Products were stabilized using NaBH(3)CN. Polyclonal antibodies were produced from rabbits after four immunizations with gossypol-LPH. In ELISA, approximately 0.4 mu caused 50% inhibition of binding (I(50)) to gossypol-BSA solid-phase, when gossypol derivatives were used competitively. This antibody showed higher affinity to lysine-derivatized gossypol, hemigossypol, gossypolone, 6, 6'-dimethoxygossypol, and hexamethoxygossypol compared to underivatized counterparts, and this antibody did not recognize naphthalene or naphthol. The ELISA results for acetone-extractable gossypol in cottonseed products (derivatized with L-(+)-lysine) showed good correlation (r(2) = 0.96) with free gossypol results obtained using the official AOCS method.     

Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity

Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.     

Effects of ionic strength on the enzymatic hydrolysis of diluted and concentrated whey protein isolate

To identify the parameters that affect enzymatic hydrolysis at high substrate concentrations, whey protein isolate (1-30% w/v) was hydrolyzed by Alcalase and Neutrase at constant enzyme-to-substrate ratio. No changes were observed in the solubility and the aggregation state of the proteins. With increasing concentration, both the hydrolysis rate and the final DH decreased, from 0.14 to 0.015 s(-1) and from 24 to 15%, respectively. The presence of 0.5 M NaCl decreased the rate of hydrolysis for low concentrations (to 0.018 s(-1) for 1% WPI), resulting in similar rates of hydrolysis for all substrate concentrations. The conductivity increase (by increasing the protein concentration, or by addition of NaCl) has significant effects on the hydrolysis kinetics, but the reason for this is not yet well understood. The results show the importance of conductivity as a factor that influences the kinetics of the hydrolysis, as well as the composition of the hydrolysates.     

Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA

Florfenicol (FF) is a broad-spectrum antibiotic used increasingly in aquaculture, livestock, and poultry to treat diseases. To avoid using labor-intensive instrumental methods to detect residues of FF in food and food products, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for florfenicol's major metabolite, florfenicol amine (FFA), was developed using a polyclonal antibody prepared in this study. FFA was covalently attached to carrier protein as immunogen by using the glutaraldehyde method. The antibodies obtained were characterized by an ELISA method and showed excellent specificity and sensitivity with the 50% inhibition values (IC 50) of 3.34 microg/L for FFA in PBS buffer. In the ELISA, sample extractions were performed by ethyl acetate/ammonium hydroxide (90 + 10, v/v) following combined acid hydrolysis of FF and its known metabolites. The limits of detection (LOD) calculated from the analysis of 20 known negative swine muscle, chicken muscle, and fish samples were 3.08, 3.3, and 3.86 microg/kg (mean + 3 SD), respectively. Recoveries of FFA fortified at the levels of 5, 50, 100, and 300 microg/kg ranged from 64.6 to 124.7%, with coefficients of variation of 11.3-25.8% over the range of FFA concentrations studied. Validation of the ELISA method with FFA-fortified swine muscle at the levels of 10, 50, 100, and 200 microg/kg was carried out using GC, resulting in a similar correlation in swine muscle ( r = 0.97). The results suggest that this ELISA is a specific, accurate, and sensitive method, which is suitable for use as a screening method to detect residues of FFA in animal edible tissues.     

Identification of angiotensin-I-converting enzyme inhibitory peptides derived from sodium caseinate hydrolysates produced by Lactobacillus helveticus NCC 2765

Angiotensin-I-converting enzyme (ACE) inhibitory activity was identified in milk proteins fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Nestle Culture Collection, Vers-chez-les-Blanc, Switzerland). Hydrolyzing sodium caseinate for 1 and 2 h inhibited ACE activity, as measured by an in vitro ACE inhibition test. The hydrolysates with the highest ACE inhibitory potential were fractionated by gel permeation chromatography and their low molecular weight fractions collected. These fractions were subsequently subfractionated by reverse-phase high-pressure liquid chromatography. Several hydrophobic subfractions showed high ACE inhibitory potential, and their peptide composition was determined using an ion trap mass spectrometer equipped with an elctrospray ionization source. Analysis of the low molecular weight fraction identified 14 peptides with known antihypertensive activity and 1 with previously described opioid activity. On the basis of the peptide composition of active subfractions, two potentially active novel sequences were defined, and the following synthetic peptides were synthesized: FVAPFPEVFG (alphaS1 39-48), ENLLRFFVAPFPEVFG (alphaS1 33-48), NENLLRFFVAPFPEVFG (alphaS1 32-48), LNENLLRFFVAPFPEVFG (alphaS1 31-48), NLHLPLPLL (beta 147-155), ENLHLPLPLL (beta 146-155), and VENLHLPLPLL (beta 145-155). The ACE inhibitory potential of these synthetic peptides was assessed, and IC50 values were determined. NLHLPLPLL (beta 147-155), which was the only synthetic peptide also present in the sodium caseinate hydrolysates, and NENLLRFFVAPFPEVFG (alphaS1 32-48) showed the highest inhibition of ACE activity, with IC50 values of 15 and 55 microM, respectively. Furthermore, the stability of all synthetic peptides was assessed using an in vitro model simulating gastric digestion. The beta-casein-derived peptides remained intact following the successive hydrolysis by pepsin and pancreatin, whereas alphaS1-casein-derived peptides were degraded by pepsin.     

Enzyme-linked immunosorbent assay development for the beta-adrenergic agonist zilpaterol

Zilpaterol is an beta-adrenergic agonist approved for use in cattle in South Africa and Mexico as a growth promoter. It is not currently approved for use in the EU, USA, or Asia. Here, we report the development of an ELISA for zilpaterol. Zilpaterol was reacted with ethyl 4-bromobutyrate followed by refluxing in 0.1 M potassium hydroxide. The resulting hapten was reacted with two carrier proteins, bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as an activating agent. Immunization of goats with the zilpaterol-butyrate-KLH resulted in an antibody useful for an ELISA. We utilized zilpaterol-butyrate-BSA as a coating antigen for ELISA development. The average IC(50) derived from the developed zilpaterol immunoassay was 3.94 +/- 0.48 ng/mL (n = 25). The antibody did not cross react with N-alkyl [bamethane, clenbuterol, (-)-isoproterenol, (+)-isoproterenol, metaproterenol, or salbutamol] or N-arylalkyl (dobutamine, fenoterol, isoxsuprine, ractopamine, or salmeterol) beta-agonists. The assay was tolerant of up to 10% (v/v) of acetone, ethanol, or methanol, and 15% (v/v) of acetonitrile or DMSO. Salt concentrations ranging from 0.05 to 1.0 M minimally affected B(0) or IC(50) values. When buffer pH was <7 or >8.8, the IC(50) values increased in comparison to those measured at pH 7.4. In conclusion, a sensitive, specific zilpaterol ELISA has been developed that can serve as a rapid screening assay.     

Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate.

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.     

Influence of EDTA and citrate on physicochemical properties of whey protein-stabilized oil-in-water emulsions containing CaCl2

The influence of chelating agents (disodium ethylenediaminetetraacetate (EDTA) and sodium citrate) on the physicochemical properties of whey protein isolate (WPI)-stabilized oil-in-water emulsions containing calcium chloride was determined. The calcium-binding characteristics of EDTA and citrate at 30 degrees C were characterized in aqueous solutions (20 mM Tris buffer, pH 7.0) by isothermal titration calorimetry (ITC). EDTA and citrate both bound calcium ions in a 1:1 ratio, but EDTA had a much higher binding constant. Oil-in-water emulsions (pH 7.0) were prepared containing 6.94% (w/v) soybean oil, 0.35% (w/v) WPI, 0.02% (w/v) sodium azide, 20 mM Tris buffer, 10 mM CaCl(2), and 0-40 mM chelating agent. The particle size, apparent viscosity, creaming stability, free calcium concentration, and particle surface potential of the emulsions were measured. The chelating agents reduced or prevented droplet aggregation in the emulsions. When they were present above a certain concentration (>3.5 mM EDTA or >5 mM citrate), droplet aggregation was prevented. The reduction of aggregation was indicated by decreases in particle size, shear-thinning behavior, apparent viscosity, and creaming. Emulsions containing chelating agents had lower free calcium concentrations and more negatively charged droplets, indicating that the chelating agents improved emulsion stability by binding calcium ions. EDTA could be used at lower concentrations than citrate because of its higher calcium ion binding constant.     

Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats

Seven kinds of angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from the hydrolysates of wakame (Undaria pinnatifida) by Protease S "Amano" (from Bacillus stearothermophilus) by using three-step high-performance liquid chromatography (HPLC) on a reverse-phase column. These peptides were identified by amino acid composition analysis, sequence analysis, and liquid chromatography-mass spectrometry (LC-MS), as Val-Tyr (IC(50) = 35.2 microM), Ile-Tyr (6.1 microM), Ala-Trp (18.8 microM), Phe-Tyr (42.3 microM), Val-Trp (3.3 microM), Ile-Trp (1.5 microM), and Leu-Trp (23.6 microM). These peptides have resistance against gastrointestinal proteases in vitro. Each peptide was determined to have an antihypertensive effect after a single oral administration in spontaneously hypertensive rats (SHR). Among them, the blood pressure significantly decreased by Val-Tyr, Ile-Tyr, Phe-Tyr, and Ile-Trp in a dose of 1 mg/kg of body weight (BW). The present study showed that antihypertensive effect in the hydrolysates of wakame by Protease S "Amano" was attributed to these peptides.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

Lipid oxidation in corn oil-in-water emulsions stabilized by casein,whey protein isolate,and soy protein isolate

Proteins can be used to produce cationic oil-in-water emulsion droplets at pH 3.0 that have high oxidative stability. This research investigated differences in the physical properties and oxidative stability of corn oil-in-water emulsions stabilized by casein, whey protein isolate (WPI), or soy protein isolate (SPI) at pH 3.0. Emulsions were prepared with 5% corn oil and 0.2-1.5% protein. Physically stable, monomodal emulsions were prepared with 1.5% casein, 1.0 or 1.5% SPI, and > or =0.5% WPI. The oxidative stability of the different protein-stabilized emulsions was in the order of casein > WPI > SPI as determined by monitoring both lipid hydroperoxide and headspace hexanal formation. The degree of positive charge on the protein-stabilized emulsion droplets was not the only factor involved in the inhibition of lipid oxidation because the charge of the emulsion droplets (WPI > casein > or = SPI) did not parallel oxidative stability. Other potential reasons for differences in oxidative stability of the protein-stabilized emulsions include differences in interfacial film thickness, protein chelating properties, and differences in free radical scavenging amino acids. This research shows that differences can be seen in the oxidative stability of protein-stabilized emulsions; however, further research is needed to determine the mechanisms for these differences.     

Effects of temperature and sodium chloride concentration on the activities of proteases and amylases in soy sauce koji

This study investigated the effects of temperature and sodium chloride concentration on the proteolytic and amylolytic activities of soy sauce koji. The optimal temperatures for both protease and amylase were found in the range of 50-55 degrees C. The protease was not stable at 55 degrees C and retained only approximately 20% residual activity after incubation at 55 degrees C for 4 h. The protease was labile in sodium chloride solution, whereas the amylase was quite stable. The residual protease activity in an 18% NaCl solution was only approximately 3%. The harvested koji was mixed with 1.5 volumes of water (v/w) and incubated at 45 degrees C for 48 h; the total nitrogen and amino nitrogen contents were 1.3 and 0.56%, respectively. The results indicated that the hydrolysis of koji at the critical temperature of 45 degrees C could be employed as a rapid fermentation method to reduce the time for soy sauce manufacturing. According to this study, the combination of 5% sodium chloride and fermentation at 45 degrees C was considered as the best condition for the prohydrolysis of koji for making soy sauce. In addition, the critical temperature of 45 degrees C was very important when used in the preparation of protein hydrolysates for the flavoring industry and for the preparation of biologically active peptides.     

Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.