Identification of food-derived elastin peptide, prolyl-glycine (Pro-Gly), in human blood after ingestion of elastin hydrolysate

Elastin hydrolysate has apparent beneficial effects, and the food-derived peptide prolyl-glycine (Pro-Gly) is present in human blood after oral ingestion. Following ingestion of elastin hydrolysate (10 g/60 kg body weight) by healthy human volunteers, peripheral blood was used to prepare plasma samples from which peptides were extracted by solid phase extraction and fractionated by size-exclusion chromatography (SEC). Peptides in the SEC fractions were derivatized with phenyl isothiocyanate (PITC) and resolved by reversed phase (RP)-HPLC. Pro-Gly was the major food-derived elastin peptide, reaching a maximum (18 μM) at 30 min after ingestion, and decreasing to approximately 20% at 4 h after ingestion. Finally, in cell culture, levels of Pro-Gly in the medium above 0.1 μg/mL significantly enhanced elastin synthesis of normal human dermal fibroblasts (NHDF) without affecting the rate of cell proliferation.     

Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates

In the present study, we identified several food-derived collagen peptides in human blood after oral ingestion of some gelatin hydrolysates. Healthy human volunteers ingested the gelatin hydrolysates (9.4-23 g) from porcine skin, chicken feet, and cartilage after 12 h of fasting. Negligible amounts of the peptide form of hydroxyproline (Hyp) were observed in human blood before the ingestion. After the oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20-60 nmol/mL of plasma) after 1-2 h and then decreased to half of the maximum level at 4 h after the ingestion. Major constituents of food-derived collagen peptides in human serum and plasma were identified as Pro-Hyp. In addition, small but significant amounts of Ala-Hyp, Ala-Hyp-Gly, Pro-Hyp-Gly, Leu-Hyp, Ile-Hyp, and Phe-Hyp were contained.     

Food-derived peptides stimulate mucin secretion and gene expression in intestinal cells

In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells.     

Comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources

We compared quantity and structures of food-derived gelatin hydrolysates in human blood from three sources of type I collagen in a single blind crossover study. Five healthy male volunteers ingested type I gelatin hydrolysates from fish scale, fish skin, or porcine skin after 12 h of fasting. Amounts of free form Hyp and Hyp-containing peptide were measured over a 24-h period. Hyp-containing peptides comprised approximately 30% of all detected Hyp. The total area under the concentration-time curve of the fish scale group was significantly higher than that of the porcine skin group. Pro-Hyp was a major constituent of Hyp-containing peptides. Ala-Hyp, Leu-Hyp, Ile-Hyp, Phe-Hyp, and Pro-Hyp-Gly were detected only with fish scale or fish skin gelatin hydrolysates. Ala-Hyp-Gly and Ser-Hyp-Gly were detected only with fish scale gelatin hydrolysate. The quantity and structure of Hyp-containing peptides in human blood after oral administration of gelatin hydrolysate depends on the gelatin source.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE

A lung extract rich in angiotensin converting enzyme (ACE) and pure ACE were immobilized by reaction with the activated support 4 BCL glyoxyl-agarose. These immobilized ACE derivatives were used for purification of ACE inhibitory peptides by affinity chromatography. The immobilized lung extract was used to purify inhibitory peptides from sunflower and rapeseed protein hydrolysates that had been obtained by treatment of protein isolates with alcalase. The ACE binding peptides that were retained by the derivatives were specifically released by treatment with the ACE inhibitor captopril and further purified by reverse-phase C18 HPLC chromatography. Inhibitory peptides with IC50 50 and 150 times lower than those of the original sunflower and rapeseed hydrolysates, respectively, were obtained. The derivative prepared using pure ACE was used for purification of ACE inhibitory peptides from the same type of sunflower protein hydrolysate. ACE binding peptides were released from the ACE-agarose derivatives by treatment with 1 M NaCl and had an IC50 a little higher than those obtained using immobilized extract and elution with captopril. Affinity chromatography facilitated the purification of ACE inhibitory peptides and potentially other bioactive peptides present in food proteins.     

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.     

Reactivity of fish and microbial transglutaminases on glutaminyl sites of peptides derived from threadfin bream myosin

Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.     

Evidence of peptides in low-molecular-weight fractions of soil organic matter

Summary Organic matter was extracted from three soils, a Berwick sandy loam, a Franklin loamy sand, and a Cumberland silty loam. The extracts were separated into high (>8000 daltons) and low-molecular-weight (<8000 daltons) fractions using gel filtration. Reverse-phase high performance liquid chromatography at 214 nm was used to separate the peptides into low-molecular-weight fractions. Peptide samples were collected with an integrated fraction collector and hydolyzed with an immobilized protease column reactor. High performance liquid chromatography with fluorescence detection was used to determine the amino-acid contents of the collected samples. The results indicated that peptide intermediates are present in soil size fractions. Greater quantities of several amino acids were released from the peptide hydrolyzates of the Berwick sandy loam and Franklin loamy sand, compared with the Cumberland silty loam, an uncultivated soil. These findings indicate that organic intermediates (e.g., peptides) are more prevalent in biologically active soils than in relatively inert soils.     

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.     

Interactions between bovine beta-lactoglobulin and peptides under different physicochemical conditions

The aim of this study was to determine if peptides could interact with beta-lactoglobulin (beta-LG) and what the physicochemical conditions promoting their interaction with the protein are. The binding of negatively charged (beta-LG 125-135 and 130-135), positively charged (beta-LG 69-83 and 146-149), and hydrophobic (alphaS1-CN 23-34 and beta-LG 102-105, both bioactive peptides) peptides to bovine beta-LG was determined using an ultrafiltration method under different physicochemical conditions: pH 3.0, 6.8, and 8.0; buffers of 0.05 and 0.1 M; 4, 25, and 40 degrees C; beta-LG/peptide ratios of 1:5 and 1:10. At pH 3.0, none of the peptides interacted with beta-LG at any temperature, buffer molarity, or beta-LG/peptide ratio probably due to electrostatic repulsions between the highly protonated species. At pH 6.8 and 8.0, charged peptides beta-LG 130-135, 69-83, and 146-149 bound to beta-LG under some physicochemical conditions, possibly by nonspecific binding. However, both hydrophobic peptides probably bind to the inner cavity (beta-barrel) of beta-LG, provoking the release of materials absorbing at 214 nm. Given the known biological activities of the hydrophobic peptides used in this study (opioid and ACE-inhibitory activities), their binding to beta-LG may be relevant to a better understanding of the physiological function of the protein.     

Development of continuous type apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in protein hydrolysates

An apparatus for continuous fractionation of peptides on the basis of amphoteric nature of sample peptides was developed. A tank (66.5 cm x 8 cm x 8 cm, L x W x H) was divided into 12 compartments by a thin agarose gel layer. A drain tube (5.5 cm in length and 0.7 cm in i.d.) was fixed through the bottom of each compartment to give a height of 4 cm from the bottom. The tank with 12 compartments and electrodes was referred to as an autofocusing unit. The peptide solution or water was delivered to the sample compartments of the first unit. The solutions drained from the first unit were successively delivered to the second and third units. To the electrodes of three units, a direct electric current was applied. By using the present apparatus, peptides in casein hydrolysate can be continuously fractionated at least for 5 h. Better resolution was obtained in the second and third units.     

Fractionation of beta-lactoglobulin tryptic peptides by ampholyte-free isoelectric focusing.

Solutions of tryptic hydrolysate of bovine beta-lactoglobulin were fractionated by liquid-phase IEF in a preparative Rotofor cell at constant power for 2 h without ampholytes in order to identify interactions between peptides. The 20 peptide fractions collected were analyzed by capillary electrophoresis and SDS-PAGE under native, denaturing, and reducing conditions. The hydrolysate was shown to be composed mainly of acidic peptides (pI 2-5, 62%) of molecular mass below 6 kDa, and numerous disulfide bonds were detected. Purified peptides (beta-LG 15-20, 71-75, 76-82, and 84-91) were also focused individually and in mixtures and matched to components of the IEF fractions obtained from the tryptic hydrolysate of beta-LG. The separation of acidic (beta-LG 84-91) and basic (beta-LG 76-82) peptides was achieved by IEF, whereas uncharged peptides (beta-LG 15-20 and 71-75) were poorly separated due to their low electrophoretic mobility. Because no peptide-peptide interaction could be identified by IEF fractionation, it is suggested that electrical fields may decrease electrostatic interactions between charged peptides.     

Accumulation and identification of angiotensin-converting enzyme inhibitory peptides from wheat germ

The incubation conditions of wheat germ for angiotensin I-converting enzyme inhibitory activity (ACEI) elevation and peptide accumulation were investigated, and five ACE inhibitory peptides were obtained. The effect of individual factors such as incubation time, temperature, initial pH, and liquid to solid ratio on ACEI and peptide concentration of incubation medium was evaluated, respectively. The combinations of four factors were further optimized using a Box-Behnken design. Under the best incubation condition (pH 4.4 with a liquid to solid ratio 8.14 mL/g at temperature 47 °C, for 7 h), maximum ACEI (92.16%) and peptide concentration (88.12 mg/g) were obtained, which were 6.2- and 2.4-fold, respectively, as compared to the unincubated wheat germ. After they were purified, five ACE inhibitory peptides, VEV, W, NPPSV, QV, and AMY, were identified by liquid chromatography/tandem mass spectrometry. The IC(50) were 115.20, 94.87, 40.56, 26.82, and 5.86 μM, respectively.     

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.     

Identification of strong aggregating regions in soy glycinin upon enzymatic hydrolysis

Upon hydrolysis with chymotrypsin, soy glycinin has a strong tendency to aggregate. The regions of glycinin from which the aggregating peptides originate were identified by accumulative-quantitative peptide mapping. To this end, the aggregating peptides were further hydrolyzed with trypsin to obtain peptides of which the sequence can be identified using RP-HPLC-MS/MS. This resulted in a hydrolysate in which 90% of the proteinaceous material was dissolved. The soluble fraction was analyzed using the method of accumulative-quantitative peptide mapping: fractionation using ion exchange chromatography, followed by identification of peptides by RP-HPLC-MS/MS, quantification based on the absorbance at 214 nm, and finally peptide mapping. For the peptide mapping the proportions in which each of the five glycinin subunits are present, as determined by Edman degradation, were taken into account. The results showed that mainly the basic polypeptide and a part of the acidic polypeptide, close to the location of the disulfide bridge between the basic and acidic polypeptides, are present in the aggregating peptide fraction. On the basis of the results obtained, an aggregation mechanism was proposed. The hydrophilic acidic polypeptides shield the hydrophobic basic polypeptides, and the former are preferentially degraded upon hydrolysis. This results in a net increase in hydrophobicity of the remaining material, which mainly consists of the basic polypeptide fragments. This increase in hydrophobicity is proposed to be the driving force in the aggregation of chymotrypsin-derived peptides of glycinin.     

Quantitative structure-activity relationship study of bitter peptides

A database consisting of 224 di- to tetradecapeptides and five amino acids was compiled to study quantitative structure-activity relationships of bitter peptides. Partial least-squares regression-1 analysis was conducted using the amino acid three z-scores and/or three parameters (total hydrophobicity, residue number, and log mass values) as X-variables and bitterness values (log 1/T where T is the bitterness threshold) as Y-variables. Using the three parameters only, significant models (p < 0.001) were obtained describing the entire data set as well as data subsets, except that comprised only of octa- to tetradecapeptides. For data sets comprising different peptide lengths, the models were improved by including the three z-scores at the N-terminal and C-terminal positions. Correlation coefficients for bitterness prediction of 48 dipeptides and 12 pentapeptides were 0.75 (RMSEP = 0.53) and 0.90 (RMSEP = 0.48), respectively. Bulky hydrophobic amino acids at the C terminus and bulky basic amino acids at the N terminus were highly correlated to bitterness.     

Investigation on the formation and the determination of gamma-glutamyl-beta-alanylhistidine and related isopeptide in the macromolecular fraction of beef soup stock

To confirm the formation of gamma-glutamyl-beta-alanylhistidine and related peptide, a model solution (amide-containing amino acids and carnosine) has been heated, and the products are investigated. Spectroscopical analysis indicates that the major product from asparagine and carnosine is beta-aspartyl-beta-alanylhistidine, and that from glutamine and carnosine is gamma-glutamyl-beta-alanylhistidine. Furthermore, to confirm the increase of the above peptides during the heating process of food, an HPLC method for the determination of these isopeptides in food protein is constructed. The isopeptides are liberated by proteolytic digestion and fractionated by solid-phase extraction using Toyopack IC-SP cartridges. The fraction containing the isopeptides is derivatized with phenylisothiocyanate (PITC) and separated and quantified by HPLC using an octadecyl-silica column. As a result of quantification, an increase of the gamma-glutamyl-beta-alanylhistidine isopeptide in the macromolecular fraction of heated beef soup stock solution has been observed. These results suggest that the formation of the isopeptide occurs in the heating of various foods containing carnosine.     

Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.