Mutation analysis of the interaction of B-type cytochrome c oxidase with its natural substrate cytochrome c-551

Heme-copper oxidases in the respiratory chain are classified into three subfamilies: A-, B- and C-types. Cytochrome bo₃-type cytochrome c oxidase from thermophilic Bacillus is a B-type oxidase that is thought to interact with cytochrome c through hydrophobic interactions. This is in contrast to A-type oxidases, which bind cytochrome c molecules primarily through electrostatic forces between acidic residues in the oxidase subunit II and basic residues within cytochromes. In order to investigate the substrate-binding site in cytochrome bo₃, eight acidic residues in subunit II were mutated to corresponding neutral residues and enzymatic activity was measured using cytochrome c-551 from closely related Bacillus PS3. The mutation of E116, located at the interface to subunit I, decreased the k_cat value most prominently without affecting the K_m value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the Cu_A site, largely affected both values, suggesting that it is important for both electron transfer and substrate binding. The mutation of D49 and E84 did not affect enzyme kinetic parameters, but the mutation of E64, E66 and E68 lowered the affinity of cytochrome bo₃ for cytochrome c-551 without affecting the k_cat value. These three residues are located at the front of the hydrophilic globular domain and distant from the Cu_A site, suggesting that these amino acids compose an acidic patch for a second substrate-binding site. This is the first report on site-directed mutagenesis experiments of a B-type heme-copper oxidase.     

THE EFFECT OF pH ON RAPESEED GLOBULIN (CRUCIFERIN) BINDING TO ANILINONAPHTHALENE-8-SULFONATE

The fluorescence emission intensity from rapeseed globulin (cruciferin) increased in the presence of anilinonaphthalene-8-sulfonate (ANS) at pH 2.0 but not at pH 10. Fluorescence titration studies showed that at pH 7 cruciferin binds 22 (±0.6) moles of ANS per mole of protein with an average dissociation constant (K_d) of 1.9 (±0.1) × 10^?5 M. At pH 2.0 the number of ligand binding sites (n) decreased to 14 (±0.2) moles of ANS bound per mole of cruciferin. However, the ANS binding affinity increased by about five times (K_d= 3.6 (±1.1) × 10^?6 M). The fluorescence emission spectrum maxima (Λ_max) for the cruciferin-ANS complex showed a blue shift at pH 2 when compared to Λ_max values at pH 7–10. These results are consistent with a loss of the quaternary and tertiary structures of cruciferin and the exposure of surface hydrophobic ANS binding sites at low pH. Cruciferin-ANS binding parameters at pH 10 were not significantly different from values at pH 7; n = 22 and K_d= 2.7 (±0.2) × 10^?5 M. Based on these ANS fluorescence measurements cruciferin is stable under alkaline conditions.     

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3 mg of N/mL of inoculum) and incubated with 10 mL of ruminal inoculum and 5 mL of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t = 0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (k_d) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters K_m (Michaelis constant) and k_d (Vmax/K_m), where Vmax = maximum velocity, were estimated directly; k_d values were adjusted (Ak_d) for the fraction FD0 using the equation Ak_d = k_d − FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean = 0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides.     

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin,trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (K _i) of which for inhibition of PepO was 60 μM. This peptide also inhibited (K _i=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (K _i=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aK _i of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.     

Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats

Oyster (Crassostrea talienwhanensis Crosse) proteins were produced from fresh oyster and subsequently digested with pepsin. The separations were performed with a Sephadex LH-20 gel filtration chromatography and a RP-HPLC. A purified peptide with sequence Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (VVYPWTQRF) was firstly isolated and characterized from oyster protein hydrolysate and its ACE inhibitory activity was determined with IC₅₀ value of 66 μmol/L in vitro. Stability study for ACE inhibitory activity showed that the isolated nonapeptide had the good heat and pH stability and strong enzyme-resistant properties against gastrointestinal proteases. Kinetic experiments demonstrated that inhibitory kinetic mechanism of this peptide was non-competitive and its K_m and K_i values were calculated. The yield of this peptide from oyster proteins was 8.5%. Furthermore, the oyster protein hydrolysate (fraction II), prepared by pepsin treatment firstly exhibited antihypertensive activity when it was orally administered to spontaneously hypertensive rat (SHR) at a dose of 20 mg/kg. These results demonstrated that the hydrolysate from oyster proteins prepared by pepsin treatment could serve as a source of peptides with antihypertensive activity.     

Purification and characterisation of a zinc-binding peptide from oyster protein hydrolysate

A novel zinc-binding peptide produced from oyster protein hydrolysis using pepsin was purified and characterised. The hydrolysate was fractionated by immobilised metal ion affinity chromatography (IMAC-Zn²⁺). The zinc-binding peptide identified by reverse-phase high-pressure liquid chromatography (RP-HPLC) and sequenced by liquid chromatography (LC/LTQ) mass spectrometry (sequence from N to C terminal) had a molecular weight of 1882.0 Da. The zinc-binding capacity of the peptide (HLRQEEKEEVTVGSLK) was 6.56 μg mg^?1 and it was preserved at 85.98% of its original level upon in vitro simulated digestion. The UV–vis and FTIR spectra demonstrate that the amino nitrogen atoms and the oxygen atoms belonging to the carboxylate groups are the primary binding sites for Zn²⁺. The results provide a feasible approach to isolate zinc-binding peptides and contribute to clarification of binding mechanism between zinc and peptides.     

Screening of peptides with a high affinity to bile acids using peptide arrays and a computational analysis

Bile acid binding peptides have attracted attention for the improvement and prevention of hypercholesterolemia. In this study, screening of bile acid high affinity peptides was investigated using computationally-assisted peptide array analysis. Starting with the screening data obtained from a limited, random 6-mer library (2212 sequences), the peptides with a high affinity to bile acid were characterized by comparison of high- and low-affinity peptides using fuzzy neural network (FNN) analysis. The physical properties of amino acids at specific positions that contribute to bile acid binding activity were extracted as the structural rule; optimization was carried out using three repeated screening cycles of the rule extraction. The extracted structural rule indicates that Trp, Tyr, Phe, Leu, Ile and Val are enriched in bile acid binding peptides. The yields of bile acid binding peptides with an affinity of above the VAWWMY peptide (soystatin, control sequence) were significantly higher in the optimized structural rule (32.5%) compared to that of the random library (3.1%), and 6 peptides were obtained with above 2.0-fold increased binding activity.     

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.     

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.     

Isolation of immunoglobulin in yolk (IgY) and rabbit serum immunoglobulin G (IgG) specific against bovine lactoferrin by immunoaffinity chromatography

Hens were intramuscularly immunized and rabbits were subcutaneously immunized once every two weeks for 6 weeks using bovine lactoferrin (LF) as antigen. Antibody titers of both yolk (IgY) and rabbit serum (IgG) were as high as 1.68×10⁸ at the 6th and 8th weeks, respectively, after the initial immunization treatment. However, antibody titer against LF in yolk was 9.4×10⁷ at 16 weeks. While antibody titer of rabbit serum declined sharply to 2.1×10⁷ at the 12th week and to 2.6×10⁶ at the 13th week after the initial immunization. The purification efficiency (specific activity of purified antibody against LF/specific activity of the corresponding antiserum or yolk against LF) of rabbit serum IgG purified by laboratory-prepared LF-Sepharose 4B immunoaffinity column (0.05 mg LF/ml wet gel) was about 2400, similar to that of IgY purified by LF-Sepharose 4B immunoaffinity column. Different amounts (0–15.0 mg) of IgY purified by LF-Sepharose 4B immunoaffinity chromatography were applied to the same column to determine the binding capacity (q_m) and dissociation constant (K_d) of LF-Sepharose 4B immunoaffinity gel for IgY specific against LF. It was found that q_m was 0.81 mg IgY/ml wet gel (1.620 mg IgY/mg LF) and K_d was 6.4×10⁻⁶ M as determined by Langmuir-type adsorption isotherms.     

Phlorotannins of the edible brown seaweed Ecklonia cava Kjellman induce sleep via positive allosteric modulation of gamma-aminobutyric acid type A-benzodiazepine receptor: A novel neurological activity of seaweed polyphenols

The primary objective was to investigate whether seaweeds have hypnotic activity. Methanol extracts of 30 seaweeds were screened for their binding activity at the GABA type A-benzodiazepine (GABA_A-BZD) receptor, a well-characterised molecular target for sedative-hypnotics. The most active seaweed was Ecklonia cava Kjellman (ECK). An ethanol extract of ECK (ECK-E) significantly potentiated pentobarbital-induced sleep in mice. In four solvent fractions separated from ECK-E, hypnotic activity was proportional to contents of total phenols and total phlorotannins, known as seaweed polyphenols. Major phlorotannins of the ethyl acetate (EtOAc) fraction with the highest activity were eckol, eckstolonol, dieckol, and triphlorethol-A, and their K_i (binding affinity, μM) values for [³H]-flumazenil binding were 1.070, 1.491, 3.072, and 4.419, respectively. Hypnotic effects of ECK-E and the EtOAc fraction were fully inhibited by flumazenil, a specific GABA_A-BZD receptor antagonist. These results imply that phlorotannins of ECK induce sleep by positive allosteric modulation of the GABA_A-BZD receptor.     

Effects of cationic property on the in vitro antioxidant activities of pea protein hydrolysate fractions

Yellow pea seed protein-derived peptides were produced through enzymatic hydrolysis of pea protein isolate, which was followed by ultrafiltration to isolate peptides with < 3 kDa sizes (PPH). The PPH was separated by cation-exchange chromatography into five peptide fractions (F1-F5) based on their affinity for the stationary phase. F1 contained peptides with the least amount (7.5%) of cationic amino acids while F5 contained peptides with more than 50% content of cationic amino acids. F1-F5 peptide fractions were evaluated for in vitro antioxidant activities in comparison to glutathione (GSH), an endogenous antioxidant peptide. Results showed that the peptide fraction with the least cationic property (F1) had significantly strongest (p < 0.05) scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radical and hydrogen peroxide (H₂O₂) when compared to F2-F5. Generally, the scavenging of O₂?B⁻ and H₂O₂ was negatively related with the cationic property of the peptide fractions. Fractionation according to cationic property resulted in significantly increased (p < 0.05) O₂?B⁻ scavenging power of the peptide fractions (F1-F5) when compared to the unfractionated PPH. The peptide fractions showed weak or nil ferric reducing power and, regardless of cationic property, were significantly (p < 0.05) more effective at inhibiting lipid oxidation than GSH over a 7-day period. In contrast, with the exception of H₂O₂ scavenging, GSH was a more effective antioxidant than PPH and the peptide fractions. We concluded that the PPH and some of its fractions have the potential to be used as therapeutic agents against chronic diseases that develop from oxidative damage, and that the cationic property of amino acids contributed negatively to the antioxidant activity of the peptide fractions.     

Biochemical characteristics of Trametes multicolor pyranose oxidase and Aspergillus niger glucose oxidase and implications for their functionality in wheat flour dough

Similar to glucose oxidase (GO), pyranose oxidase (P₂O) may well have desired functionalities in some food applications in general, particularly breadmaking. As its name implies, P₂O oxidises a variety of monosaccharides. P₂O purified from a culture of Trametes multicolor (P₂O-Tm) had high affinity towards d-glucose (K_M = 3.1 mM) and lower affinity to other monosaccharides. GO from Aspergillusniger (GO-An) had a K_M value of 225 mM towards glucose, which points to a significant difference in glucose affinity between the two enzymes. Furthermore, P₂O-Tm had higher affinity towards O₂ (K_M = 0.46 mM) than GO-An (K_M = 2.9 mM). Dehydroascorbic acid did not accept electrons in the reactions catalysed by P₂O-Tm and GO-An. For the same activity towards glucose in saturating conditions, the rate of ferulic acid oxidation in a model system and of thiol oxidation in a wheat flour extract were higher with P₂O-Tm, than with GO-An. The demonstrated differences in properties and functional features between P₂O-Tm and GO-An allow prediction of differences in functional behaviour of the enzymes, in food applications.     

Antigenicity of β-lactoglobulin reduced by combining with oleic acid during dynamic high-pressure microfluidization: Multi-spectroscopy and molecule dynamics simulation analysis

Some food components can modulate the antigenicity of β-lactoglobulin (β-LG). This study investigated the role of oleic acid (OA) in reducing the antigenicity of β-LG. The results indicate the antigenicity of β-LG gradually decreased from 15 (sample with no OA) to 9.86, 7.51, and 6.01 µg/mL when interacting with OA during dynamic high-pressure microfluidization treatment at 0.1, 80, and 160 MPa. Although binding sites (n) of β-LG combined with OA at 0.1, 80, and 160 MPa decreased from 0.79 to 0.5 and 0.66, β-LG had a higher binding affinity (K_a) to OA than that of untreated β-LG. The values of K_a for β-LG/OA at 0.1, 80, and 160 MPa were 5.51 × 10⁶, 17.43 × 10⁶, and 49.75 × 10⁶M^?1, respectively. The molecule dynamic simulation showed that the OA molecules located at both β-barrel (site 1) interacted with Lys60, Glu62, and Lys69 and outer surface site 2 consisting of Tyr20, Tyr42, Ser21, Glu157, and His161. Additionally, when binding with OA during the dynamic high-pressure microfluidization treatment, the conformation of β-LG changed, reflected by the decrease of fluorescence intensity and total sulfhydryl group content, the increase of surface sulfhydryl group content, and secondary structure changes of β-LG. These results deduce that some epitopes may be masked by OA or modified by the conformational changes, resulting in the decline of antigenicity of β-LG molecules.     

Purification and characterisation of angiotensin I converting enzyme inhibitory peptides from lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F₇ (from papain-trypsin hydrolysate), F₈ (from papain hydrolysate) and F₃ (from trypsin hydrolysate) fractions. The IC₅₀ values were 0.03, 0.155 and 0.23 mg/ml for F₇, F₈ and F₃, respectively. The F₇ fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver-Burk plots suggest that the F₇ peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (K_m, V_max, and K_i) for the F₇ peptide were measured and compared to the control.     

Antioxidant peptides isolated from synbiotic yoghurt exhibit antiproliferative activities against HT-29 colon cancer cells

Milk proteins are considered a reservoir of peptides possessing various bioactivities. Using ultrafiltration followed by reversed phase-high performance liquid chromatography, antioxidant peptides were purified from crude peptide extract of probiotic yoghurt supplemented with pineapple peel powder stored for 28 days at 4 °C. Two β-casein-derived peptides, ¹⁹³YQEPVLGPVRGPFPIIV²⁰⁹ and ⁶⁹SLPQNIPPLTQTPVVVPPF⁸⁷ (designated P17 and P19, respectively), were identified and their antioxidant and anticancer activities assessed. P17 showed high scavenging activity against 2,2′-azino-bis(3-ethyl benzothiazoline-6-sulphonic acid) radicals, with an IC₅₀ of 29.88 μg mL⁻¹, compared with P19 (IC₅₀ 1.44 mg mL⁻¹). Furthermore, the proliferation of HT-29 colon cancer cell line was inhibited (41.49% and 38.55%, respectively, by P17 and P19 at 3 mg mL⁻¹) via inducing apoptosis and cell cycle arrest in G₂/M-phase. In vitro gastrointestinal digestion of peptides resulted in increased bioactivities. These findings indicate a potential for utilising both of these peptides to manage oxidative stress mediated diseases and disorders including cancers.     

Binding of vitamin A with milk α- and β-caseins

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV–visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K_{retinol-α-caseins} = 1.21 (±0.4) × 10⁵ M⁻¹ and K_{retinol-β-caseins} = 1.11 (±0.5) × 10⁵ M⁻¹ and K_{retinoic acid-α-caseins} = 6.2 (±0.6) × 10⁴ M⁻¹ and K_{retinoic acid-β-caseins} = 6.3 (±0.6) × 10⁴ M⁻¹. The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid–casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules.     

New insights into the cholesterol esterase- and lipase-inhibiting potential of bioactive peptides from camel whey hydrolysates: Identification,characterization, and molecular interaction

Novel antihypercholesterolemic bioactive peptides (BAP) from peptic camel whey protein hydrolysates (CWPH) were generated at different time, temperature, and enzyme concentration (%). Hydrolysates showed higher pancreatic lipase- (PL; except 3 CWPH) and cholesterol esterase (CE)-inhibiting potential, as depicted by lower half-maximal inhibitory concentration values (IC₅₀ values) compared with nonhydrolyzed camel whey proteins (CWP). Peptide sequencing and in silico data depicted that most BAP from CWPH could bind active site of PL, whereas as only 3 peptides could bind the active site of CE. Based on higher number of reactive residues in the BAP and greater number of substrate binding sites, FCCLGPVPP was identified as a potential CE-inhibitory peptide, and PAGNFLPPVAAAPVM, MLPLMLPFTMGY, and LRFPL were identified as PL inhibitors. Molecular docking of selected peptides showed hydrophilic and hydrophobic interactions between peptides and target enzymes. Thus, peptides derived from CWPH warrant further investigation as potential candidates for adjunct therapy for hypercholesterolemia.     

Peptide Microencapsulation by Core–Shell Printing Technology for Edible Film Application

This paper presents a new microencapsulation methodology for incorporation of functional ingredients in edible films. Core–shell microcapsules filled with demineralized water (C) or 1 % (w/v) peptide solution (Cp) were prepared using the microencapsulation printer technology. Shell material, composed of a stearic acid/carnauba wax mixture (75:25), represented around 10 % of the capsule weight, corresponding to a shell material/peptide ratio of 13.3:1 on dry basis. C capsules were more spherical and homogeneous than Cp ones. Cp’s more irregular morphology would explain the slightly higher size of d ₉₀ (126 μm) compared to C (122.50 μm). Cp microcapsules were more stable at pH 5 and 7 (<30 % peptide released in 3 h) than at pH 2 and 9.2 (40–50 % released in 3 h). A procedure for homogeneous microencapsule inclusion in hydrophilic Laminaria digitata edible films was developed, without losing microcapsule integrity either in the filmogenic solution or during the drying process. Films with added microcapsules were stronger and more deformable, more opaque, more water-soluble but less permeable to water vapour and less resistant to perforation.     

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC₅₀; <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α_S1-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity.