Proteolytic Hydrolysates Research Articles

Chemical and Sensory Evaluation of Umami Taste Derived from Proteolytic Hydrolysate of Pila ampullacea

In this study, umami taste was produced and identified from bromelain and trypsin hydrolysate of Pila ampullacea (PA). The PA hydrolysates, obtained from individually bromelain and trypsin with various Enzyme-to-Substrate ratio (E/S) of 1/10, 1/20, and 1/100 (w/v) using divers proteolysis times (3, 6, 9, 12, 15 and 18 h), were evaluated their chemical properties by degree of hydrolysis (DH), total peptide content, and amino acid content. Two chosen proteolysis conditions of PA hydrolysates were concluded by sensory evaluation using hedonic test and principal component analysis (PCA). PA hydrolysate using bromelain with E/S of 1:10 (w/v) for 18 h was one of the 2 chosen proteolysis conditions, which had DH value of 56.56 ± 1.65 %, total peptide content of 10.89 ± 0.09 mg/mL, and amino acid content of 95.34 ± 0.12 ppm. On the other hand, the chosen from trypsin digestion used E/S of 1:10 (w/v) for 15 h, which had DH value of 49.71 ± 0.22 %, total peptide content of 6.44 ± 0.28 mg/mL, and amino acid content of 81.43 ± 1.29 ppm. PA hydrolysates were subjected to explain the released peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database-assisted identification. There were many identified peptides contained in PA hydrolysates that contributed to umami taste. Their sequences from bromelain digestion were GPEGPQGPPGPRG, GIMLGAA, GLPGLPGLKGDPGEPGLP, GKDGEAG, GLVMDSCAGH, and EEKITEDDDAVGDDAENR. Several peptide fragments from trypsin digestion were GQTVIGL, GLPGLPGLSGPKG, DTGPAGPAGPAGPQGPR and QTLEKALSHVIQEFETEKQLITVNAR. HIGHLIGHTS Umami taste derived from proteolytic hydrolysate of Pila ampullacea was firstly reported The findings revealed that Pila ampullacea hydrolysates using bromelain with the Enzyme-to-Substrate ratio (E/S) 1:10 (w/v) for 18 h and trypsin with E/S of 1:10 (w/v) for 15 h had exhibited as the proficient treatment for hydrolysis conditions and selected based on sensory properties This new method will be of considerable advantage to the best practice of umami taste establishment as flavor enhancer in the food manufacture GRAPHICAL ABSTRACT

OxLDL-Induced Foam Cell Formation Inhibitory Activity of Pepsin Hydrolysate of Ark Shell (Scapharca subcrenata (Lischke, 1869)) in RAW264.7 Macrophages

Inhibitory effect of ark shell (Scapharca subcrenata (Lischke, 1869)) proteolytic hydrolysates (ASHs) on oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation was investigated. Two types of ASHs were prepared by Alcalase® and pepsin, ASAH (ark shell-Alcalase® hydrolysates), and ASPH (ark shell-pepsin hydrolysate). Oil Red O staining results showed that ASPH suppressed foam cell formation and lipid accumulation more than ASAH in oxLDL-induced foam cell formation of RAW264.7 macrophages. ASPH reduced the levels of total cholesterol, cholesterol ester, and free cholesterol in oxLDL-treated RAW264.7 macrophages. It was found that ASPH increased cholesterol efflux and decreased cholesterol influx rate. In this regard, protein expressions of CD36 and scavenger receptor class A1 (SR-A1) for cholesterol influx and ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) for cholesterol efflux were investigated. ASPH treatment resulted in an increase of ABCA1 and ABCG1 expression but downregulated CD36 and SR-A1 expression. Furthermore, ASPH suppressed production of proinflammatory cytokines, including tumor necrosis factor-α and interleukin-6 and -1β, through regulating nuclear factor-kappa B (NF-κB) in oxLDL-induced foam cell formation of RAW264.7 macrophages. Taken together, our data indicate that ASPH might be a useful ingredient in functional foods for ameliorating atherosclerosis by preventing foam cell formation.

Comparison of Functional Properties of Blood Plasma Collected from Black Goat and Hanwoo Cattle.

Slaughterhouse blood is a by-product of animal slaughter that can be a good source of animal protein. This research purposed to examine the functional qualities of the blood plasma from Hanwoo cattle, black goat, and their hydrolysates. Part of the plasma was hydrolyzed with proteolytic enzymes (Bacillus protease, papain, thermolysin, elastase, and α-chymotrypsin) to yield bioactive peptides under optimum conditions. The levels of hydrolysates were evaluated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The antioxidant, metal-chelating, and angiotensin I-converting enzyme (ACE) inhibitory properties of intact blood plasma and selected hydrolysates were investigated. Accordingly, two plasma hydrolysates by protease (pH 6.5/55°C/3 h) and thermolysin (pH 7.5/37°C/3-6 h) were selected for analysis of their functional properties. In the oil model system, only goat blood plasma had lower levels of thiobarbituric acid reactive substances than the control. The diphenyl picrylhydrazyl radical scavenging activity was higher in cattle and goat plasma than in proteolytic hydrolysates. Ironchelating activities increased after proteolytic degradation except for protease-treated cattle blood. Copper-chelating activity was excellent in all test samples except for the original bovine plasma. As for ACE inhibition, only non-hydrolyzed goat plasma and its hydrolysates by thermolysin showed ACE inhibitory activity (9.86±5.03% and 21.77±3.74%). In conclusion, goat plasma without hydrolyzation and its hydrolysates can be a good source of bioactive compounds with functional characteristics, whereas cattle plasma has a relatively low value. Further studies on the molecular structure of these compounds are needed with more suitable enzyme combinations.

Preparation of antioxidant peptides from Moringa oleifera leaves and their protection against oxidative damage in HepG2 cells.

Moringa oleifera leaves are a kind of new food raw materials, rich in functional factors, M. oleifera leaves aqueous extract have antioxidant activity and M. oleifera leave protein is an important active ingredient in the aqueous extract. Numerous studies have shown that peptides have strong antioxidant activity. To reveal the antioxidant effects of M. oleifera (MO) leaves peptides, MO leave antioxidant peptides were isolated and prepared to clarify their antioxidant activity. MLPH1 (<1 kDa), MLPH3 (1~3 kDa), MLPH5 (3~5 kDa), and MLPH10 (5~10 kDa) fractions were obtained by the membrane ultrafiltration classification of MO leaves proteolytic hydrolysate (MLPH). MLPH1 was further separated by centrifugal filters, and the fraction separated by <1 kDa (MLPH1-1) was identified and analyzed by LC-MS/MS. The purpose of this study was to investigate the effect of MO leaves antioxidant peptide pretreatment on H2O2-treated HepG2 cells and to refine the antioxidant activity. The results showed that MLPH1 had the strongest antioxidant activity, and three MO leaves antioxidant peptides (LALPVYN, LHIAALVFQ, and FHEEDDAKLF) were obtained. The peptide with the sequence LALPVYN and a molecular weight of 788.44 Da had the strongest antioxidant activity. After 24 h of LALPVYN pretreatment, the cell viability and the CAT, GSH-Px, and SOD enzyme activity were significantly increased, and the MDA, ROS, and apoptosis rates were significantly decreased. These results provide a theoretical basis for further research on the antioxidant mechanism of MO leaves peptides.

Antioxidative Peptides from Proteolytic Hydrolysates of False Abalone (Volutharpa ampullacea perryi): Characterization, Identification, and Molecular Docking.

Antioxidative peptides were produced from false abalone (Volutharpa ampullacea perryi) using enzymatic hydrolysis. Trypsin produced the most bioactive hydrolysates with the highest scavenging ABTS+• free radicals compared to pepsin, alcalase, neutrase, and flavourzyme. The response surface methodology studies on trypsin hydrolysis indicated that the hydrolysis temperature, time, and pH were interacted with each other (p < 0.05), and the optimal conditions were hydrolysis at 51.8 °C for 4.1 h, pH 7.7 and the maximum predicted hydrolysis degree was 13.18% and ABTS+• scavenging activity of 79.42%. The optimized hydrolysate was subjected to ultrafiltration fractionation, and the fraction with MW < 3 kDa showed the highest ABTS+• scavenging activity. There were 193 peptide sequences identified from this peptide fraction and 133 of them were successfully docked onto human myeloperoxidase (MPO), an enzyme involved in forming reactive oxidants in vivo. The highest scored peptide, no. 39, consists of DTETGVPT. Its structure and molecular interactions with MPO active site were compared with previously characterized peptide hLF1-11. The interactions between peptide no. 39 and MPO include electrostatic charge, hydrogen bonds, and covalent bonds. The antioxidative peptide produced in this research may exert antioxidant activity in vivo due to its potential inhibition effect on MPO.

Antioxidant and angiotensin-I converting enzyme inhibitory peptides from Hippocampus abdominalis

Bioactive peptides isolated from edible marine sources have been used as nutraceuticals and functional foods. The present study focuses on the alcalase hydrolysate of Hippocampus abdominalis for the isolation of antioxidative and angiotensin-I converting enzyme (ACE) inhibitory peptides. Initially, the H. abdominalis alcalase hydrolysate (HA) was separated using ultrafiltration (MWCO = 5 kDa), and have obtained the fraction, HA-III (MW ≤ 5 kDa), with strong bioactivity. This was further separated using gel filtration chromatography (Sephadex G-10) and reverse-phase high-performance liquid chromatography (RP-HPLC). The active Sephadex G-10 fraction was analyzed by the Q-TOF mass spectrometry, nine peptides were identified within the molecular mass range between 757.8 and 990.1 Da. Three peptides, GIIGPSGSP, IGTGIPGIW, and QIGFIW, showed strong ACE inhibitory activity and alkyl radical-scavenging activity. Molecular docking studies revealed that the ACE inhibitory activity of the three active peptides is mainly due to hydrogen bonding interactions and active site interactions between active peptides and ACE. This study demonstrated that the active peptides derived from H. abdominalis can be isolated and the proteolytic hydrolysates of it are potential antioxidant and antihypertensive agents.

Angiotensin I-converting enzyme inhibitory peptides from an enzymatic hydrolysate of flounder fish (Paralichthys olivaceus) muscle as a potent anti-hypertensive agent

The aim of this study was to purify peptides with anti-hypertensive properties from a hydrolysate of flounder fish muscle. Among four proteolytic hydrolysates, pepsin showed the strongest angiotensin-I converting enzyme (ACE) inhibitory activity. The pepsin hydrolysate was fractionated by ultrafiltration, gel filtration chromatography and reverse-phase high performance liquid chromatography, and two novel peptides were purified. The IC50 values of the two peptides were 79μM and 105μM, respectively, and the Lineweaver–Burk plots suggested that they act as a competitive and a non-competitive inhibitor of ACE, respectively. Moreover, we predicted the 3D structure of ACE and used a molecular docking program to simulate binding between ACE and the peptides. These molecular modeling results indicated strong binding and interaction energies, and systolic blood pressures were reduced by administration of both peptides in spontaneously hypertensive rats. These results suggested that the enzymatic hydrolysate of flounder fish muscle includes novel ACE inhibitory peptides that may be beneficial as a functional food for treating hypertension.

Data in support of optimized production of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins.

VY-7 has been demonstrated as a potent ACE inhibitory peptide in the previous study [1]. In this article, we provide accompanying data about the identification of bitter melon seed proteins (BMSPs), and quantitative analysis and optimized production of VY-7 in BMSPs hydrolysate.

Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins

In this study, new angiotensin-I converting enzyme (ACE) inhibitory peptides were comprehensively identified from a thermolysin digest of bitter melon (Momordica charantia) seed proteins. The hydrolysate was fractionated by reversed-phase high performance liquid chromatography (RP-HPLC), and the inhibitory activities of the resulting fractions were evaluated using ACE inhibitory assay. Two novel ACE inhibitory peptides (VY-7 and VG-8) were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database-assisted peptide sequencing. VY-7 and VG-8 were derived from momordin A and MAP30, respectively, and their IC50 values were as low as 8.64±0.60 and 13.30±0.62μM, respectively. Lineweaver–Burk plots further indicated that VY-7, which showed the best IC50 value, acts as a competitive inhibitor. Notably, the content of VY-7 in crude thermolysin digest was determined to be as high as 14.89±0.88μg/mg using LC-MS/MS quantification. In the spontaneously hypertensive rat (SHR) model, oral administration of VY-7 at 2mg/kg body weight significantly decreased the systolic blood pressure. The interaction between VY-7 and ACE was examined using molecular docking calculations and the results suggested that certain residues of VY-7 can fit perfectly into the S1, S1’ and S2’ regions of the binding pocket of ACE. Biological significanceOne of the most common supportive therapies for treating hypertension is the use of synthetic drugs to inhibit ACE activity. Synthetic ACE inhibitors possess good antihypertensive effects, but come with accompanying side effects. Therefore, food-derived ACE inhibitory peptides are regarded as safer alternatives and are attracting much attention for hypertension treatment. In this study, we comprehensively identified peptides derived from bitter melon (Momordica charantia) seed proteins (BMSPs) using a shotgun proteomics approach. Based on results from an in vitro ACE inhibitory assay, two peptides (VY-7 and VG-8) derived from momordin A and MAP30 proteins, respectively, showed good ACE inhibitory activities. For VY-7, which showed the best IC50 value (8.64±0.60μM), the inhibition type was determined to be competitive inhibition, as found using a Lineweaver–Burk plot. The novel ACE inhibitory peptide VY-7 (at 2mg/kg body weight) as well as the crude hydrolysate of BMSPs (at 10mg/kg body weight) showed significant and moderate antihypertensive effects, respectively, in an animal model of hypertension, spontaneously hypertensive rats (SHRs). The present work demonstrated the screening of ACE inhibitory peptides from BMSPs and, as far as we know, VY-7 is the first well-characterized antihypertensive peptide derived from bitter melon seeds.

Optimization of Hydrolysis Conditions for the Production of Iron-Binding Peptides from Mackerel Processing Byproducts

The aim of this study was focused on optimization of enzymatic hydrolysis conditions for the production of iron-binding peptides from marine mackerel processing byproducts. The marine mackerel processing byproducts protein were hydrolyzed using trypsin, Protamex, Flavourzyme, Alcalase and Neutrase. Alcalase and Protamex proteolytic hydrolysates exhibited the highest iron-binding capacity; however, Alcalase proteolytic hydrolysate had higher degree of hydrolysis than that of Protamex. A four-factor-three-level composition central design experiment in response surface methodology was used to optimize the enzymatic hydrolysis conditions of Alcalase. The optimal enzymatic hydrolysis conditions were temperature of 46.0°C, time of 2.01 h, pH 8.35 and enzyme to substrate 6460 U/mL. The quadratic model predicted well about the actual measured value. The average iron-binding capacity of three verification experiment was 6.62 mg-EDTA/g-protein, which was much closed to model predicted value of 6.69 mg-EDTA/g-protein.

ACE-Inhibitory Properties of Proteolytic Hydrolysates from Giant Jellyfish Nemopilema nomurai

This study aimed to determine the degree of hydrolysis and angiotensin-I-converting enzyme (ACE)-inhibitory activity of Giant Jellyfish Nemopilema nomurai (jellyfish) hydrolysates. The degree of hydrolysis using six proteolytic enzymes (Alcalase, Flavozyme, Neutrase, papain, Protamex, and trypsin) ranged from 13.1-36.8% and the inhibitory activities from 20.46-79.58%. Using papain hydrolysate, we newly isolated and characterized ACE-inhibitory peptides with a molecular weight of 3,000-5,000 Da that originated from jellyfish collagen. The purified peptide (FII-b) was predicted to be produced from an alpha-2 fragment of the type IV collagen of jellyfish. The N-terminal sequence of FII-b was Asp-Pro-Gly-Leu-Glu-Gly-Ala-His-Gly- and showed 87% identity to the collagen type IV alpha-2 fragment of Rattus norvegicus and a predicted protein from Nematostella vectensis, indicating that the ACE-inhibitory peptide originated from the collagen hydrolysate and had an <TEX>$IC_{50}$</TEX> value of 3.8 <TEX>${\mu}g$</TEX>/mL. The primary structure of the fragment is now being studied; this peptide represents an interesting new type of ACE inhibitor and will provide knowledge of the potential applications of jellyfish components as therapies for hypertension.

쌀 시럽박의 단백질 가수분해 특성

Rice syrup meal (RSM) was enzymatically hydrolyzed using eight commercial proteases (Protamex, Neutrase, Flavourzyme, Alcalase, Protease M, Protease N, Protease A, Molsin F) for 4 hr at optimum pH and temperature. Proteolytic hydrolysates were examined in supernatant and precipitate using Lowry protein assay, semimicro Kjeldahl method and gravimetric method using weight difference before and after enzymatic hydrolysis. Although RSM contains a high amount of protein (71.2%), only a very small amount of protein was hydrolyzed. Two proteases (Protease M and Protease N) were found to be the most effective in the hydrolysis of RSM protein. In Lowry method, 57.5 and 59.0 ㎎ protein/g RSM were hydrolyzed after Protease M and Protease N treatments, respectively. In gravimetric method, 80.0 and 85.4 ㎎ protein/g RSM were hydrolyzed after Protease M and Protease N treatments. In Kjeldahl method, 67.43 and 70.43 ㎎ protein/g RSM were hydrolyzed after Protamex and Protease N treatments, respectively. For synergistic effect, two or three effective commercial proteases (Protease M, Protease N and Protease A) were applied to RSM at one time. The highest hydrolysis of RSM protein was observed in both Lowry protein assay (80.3 ㎎ protein/g RSM) and gravimetric methods (153.2 ㎎ protein/g RSM) when three commercial proteases were applied at one time, suggesting the synergistic effect of those proteases.

Milk casein-derived tripeptides, VPP and IPP induced NO production in cultured endothelial cells and endothelium-dependent relaxation of isolated aortic rings

Milk casein-derived tripeptides, valyl prolyl proline (VPP), and isoleucyl prolyl proline (IPP) inhibit angiotensin-converting enzyme (ACE) and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans. On the top of these results, we have recently reported that the hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, enforcing us to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides. For this purpose, we examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO(x) (NO(2) and NO(3)) concentration in the medium was significantly higher than that of the control. Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists. These lines of results suggested that both VPP and IPP induced production of vasodilative substances including NO.

Structure and antioxidant activity of high molecular weight Maillard reaction products from casein–glucose

Maillard reaction products (MRPs) were prepared from a casein–glucose reaction and ultrafiltrated to provide six fractions. The high molecular weight glycated proteins (melanoprotein) were further purified with a Sephadex G-75 column. Two fractions were obtained and analysed for their reducing power and Fe 2+ chelating activity. Results obtained from the first fraction, analysed by Fourier transform infrared spectroscopy (FT-IR) indicated that the amide I, II and III bands of casein were changed by the Maillard reaction. The obtained samples were also hydrolysed with pepsin and trypsin in vitro, and the proteolytic hydrolysates were evaluated for their antioxidant activities. Non-hydrolysed melanoproteins exhibited the highest reducing power, but peptic hydrolysates of different MRPs were more efficient in radical-scavenging activity.

Screening for Nutritive Peptides That Modify Cholesterol 7α-Hydroxylase Expression

Bioactive peptides with a variety of effects have been described from several nutritive proteins. They exhibit antimicrobial, blood-pressure lowering, antithrombotic, immunomodulatory, and cholesterol-modulating effects. In this study, we have examined whether peptides derived from food proteins might influence bile acid synthesis. A reporter gene cell line that carries a cholesterol 7alpha-hydroxylase promoter fragment fused to firefly luciferase ( cyp7a-luc) was used to screen for nutritive peptides affecting cyp7a expression, the enzyme catalyzing the rate-limiting step in bile acid synthesis. Proteolytic hydrolysates were prepared from soy protein and bovine casein with pepsin, trypsin, chymotrypsin, and elastase and size fractionated using ultrafiltration. Several bioactive hydrolysates could be identified that inhibited luciferase expression. Also, an activation of kinase (AKT, ERK, p38-MAPK) signaling could be observed. Selected hydrolysates were further fractionated by reversed-phase HPLC. Bioactive HPLC-fractions were obtained from casein but not from soy hydrolysates; however, activity could not be recovered in single peak fractions. Peptides in such fractions were identified by mass spectrometry. Five selected peptides from alpha S1-casein present in active fractions were synthesized, but none of these showed activity in the cyp7a-luc screening system. However, two of them activated MAP-kinase signaling similar to the hydrolysates, which suggests, that these peptides are involved in cyp7a regulation by the casein hydrolysates.

Antioxidant activity of sulphated polysaccharide conjugates from abalone (Haliotis discus hannai Ino)

The water-soluble sulphated polysaccharide conjugates were obtained from abalone viscera (Haliotis discus hannai Ino) by alkaline protease extraction followed by ethanol precipitation. Their antioxidant activities were evaluated in vitro by hydroxyl radicals scavenging activity, reducing power and metal chelating activity. Those various antioxidant activities were compared to standard antioxidants ascorbic acid and EDTA. The experimental results indicated that the crude extract having notable hydroxyl free radicals scavenging activity and moderate reducing power and chelating potency. The crude sulphated polysaccharide conjugates was enzymatically hydrolyzed by five commercially available proteases (trypsin, vernase, neutrase, pepsin and papain), and the resultant digests were tested for their antioxidant activities. Those proteolytic hydrolysates, although improving the hydroxyl radical scavenging activity in all cases except one, had lower reducing power and 3–15 times lower chelating ability than the native extract. Product derived from pepsin hydrolysate was fractionated by gel-filtration chromatography with sephadex G-100, giving two fractions containing sulphated polysaccharide conjugates termed ACP I and ACP II. The neutral monosaccharide composition of ACP I is rhamnose, fucose, xylose, mannose, galactose and glucose in a molar ratio of 1.00:45.14:4.00:5.36:33.18:2.15, with an average molecular weight of about 271 kDa. The neutral monosaccharide composition of ACP II is rhamnose, fucose, xylose, mannose, galactose and glucose in a molar ratio of 1.00:12.51:1.33:4.98:16.08:1.46, with an average molecular weight of about 6 kDa.

Allergenicity of Proteolytic Hydrolysates of the Soybean 11S Globulin

A substantial portion of the human population has immune hypersensitivities to various food materials. Soybean is one of the most common foods involved in such hypersensitivity reactions, especially in younger children. In this study, we investigated the effect of peptic and chymotryptic hydrolysis on the allergenicity of the 11S soybean globulin, which is the primary soybean allergen. The 11S globulin is composed of both acidic and basic polypeptides, and we found that the acidic polypeptide was effectively hydrolyzed, while basic polypeptide was more resistant to hydrolysis. The 11S globulin hydrolysate was size-fractionated by gel filtration, and 9 of the fractions obtained were tested for allergenicity against sera from 6 soybean-allergenic patients. The overall allergenicity of soybean 11S globulin was reduced by peptic and chymotryptic hydrolysis, although a gel filtration fraction with a major peptide of 20 kDa was highly immunoreactive. Hydrolyzed fragments of less than about 20 kDa were not immunoreactive.

Casein Hydrolysate Containing the Antihypertensive Tripeptides Val-Pro-Pro and Ile-Pro-Pro Improves Vascular Endothelial Function Independent of Blood Pressure-Lowering Effects: Contribution of the Inhibitory Action of Angiotensin-Converting Enzyme

Accumulating evidence shows that deterioration of vascular endothelial function underlies the pathophysiology of cardiovascular diseases following lifestyle-related diseases. Both Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which are tripeptides derived from proteolytic hydrolysate of milk casein, inhibit angiotensin-converting enzyme (ACE), suggesting that both VPP and IPP may improve vascular endothelial function, because many ACE inhibitors are known to improve endothelial function. We investigated the effects of ACE-inhibitory food component in humans with mild hypertension, since there has been no report on such effects. The study was conducted by the placebo-controlled, double-blind crossover method in 25 male subjects with mild hypertension. After casein hydrolysate containing both VPP and IPP were administered for 1 week, reactive hyperemia of the left upper forearm was measured using plethysmography as an index of vascular endothelial function. Since one subject dropped out, we analyzed the data of 24 subjects. The reactive hyperemia of the left upper forearm was produced by a 5 min occlusion using inflation of a cuff. The maximum blood flow during reactive hyperemia was 20.8+/-6.7 mL/min/100 mL tissue in the placebo group, whereas it increased remarkably to 30.0+/-10.4 mL/min/100 mL tissue in the group administered casein hydrolysate containing both VPP and IPP (p<0.001). There was no change in systemic blood pressure, indicating that the improvement of the vascular endothelial function attributable to VPP and IPP is independent of hemodynamic changes. We conclude that casein hydrolysate containing VPP and IPP improves the vascular endothelial dysfunction in subjects with mild hypertension. The continuous intake of VPP and IPP could help to prevent cardiovascular diseases in hypertensive subjects.

Improvement on functional properties of wheat gluten by enzymatic hydrolysis and ultrafiltration

Four fractions (100-, 50-, 20-K and permeate) from a proteolytic hydrolysate (DH=2.8%) of wheat gluten were separated using size fractionation on ultrafiltration membranes with molecular weight cut-offs of 100, 50 and 20 kDa and their functional properties evaluated. Proteolysis led to a significant ( P<0.05) increase in solubility of gluten fractions at pHs between 2–10 and a shift of the pI value from 6–7 to 5. The solubilities of 100-K, 50-K, 20-K and permeate fractions were significantly ( P<0.05) improved compared with the untreated control and the hydrolysate. The 50-K fraction was superior to other three fractions for emulsion activity index between pH 2 and 10. The most stable foam was given by the 100-K fraction which showed 65.8% of initial foam while the control sample gave only 23% of foam, after 60 min resting. Foam stability decreased as the molecular mass of hydrolysate fractions decreased. Furthermore, after proteolysis, the surface hydrophobicity ( H 0) of gluten increased significantly ( P<0.05) compared with the control except for the permeate fraction. The highest value of H 0 was given by the 100-K fraction, followed by the 50-K, 20-K and permeate fractions. In addition, proteolysis resulted in a decrease in the storage modulus of gels.

Peptides From the N-terminal End of Bovine Lactoferrin Induce Apoptosis in Human Leukemic (HL-60) Cells

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC50 value of this peptide was 6.3μg/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC50 values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.