Vascular effects of egg white‐derived peptides in resistance arteries from rats. Structure–activity relationships

BACKGROUND: The vasodilator properties of several peptide sequences derived from egg white proteins were screened in mesenteric resistance arteries from Wistar‐Kyoto rats. For this, third‐order branches of the mesenteric arteries from 6‐month‐old male rats were used. The vasodilator responses, with or without endothelium, to several peptides (0.1 mmol L^?1) were analysed in an isometric myograph. Moreover, the effect of nitric oxide (NO) synthase (L‐NAME, 100 µmol L^?1) and cyclooxygenase (indomethacin, 10 µmol L^?1) inhibitors on the vasodilator response was tested. RESULTS: The peptides Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu, Arg‐Ala‐Asp‐His‐Pro‐Phe, Arg‐Ala‐Asp‐His‐Pro, Tyr‐Arg‐Gly‐Gly‐Leu‐Glu‐Pro‐Ile‐Asn‐Phe, Arg‐Asp‐Ile‐Leu‐Asn‐Gln and Val‐Pro‐Pro showed a high endothelium‐dependent vasorelaxation, whereas Phe‐Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu was only partially endothelium‐dependent. The relaxation induced by Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu, Arg‐Ala‐Asp‐His‐Pro‐Phe, Arg‐Ala‐Asp‐His‐Pro, Arg‐Asp‐Ile‐Leu‐Asn‐Gln and Val‐Pro‐Pro was mainly mediated by NO, since the response was inhibited only by L‐NAME, while both L‐NAME and indomethacin inhibited the vasodilator response induced by Phe‐Arg‐Ala‐Asp‐His‐Pro‐Phe‐Leu and Tyr‐Arg‐Gly‐Gly‐Leu‐Glu‐Pro‐Ile‐Asn‐Phe. The presence of Arg or Tyr at the N‐terminal position could be related to the vasodilator activity of these compounds in this vascular bed. The well‐known angiotensin‐converting enzyme inhibitor captopril showed only a slight vasodilator effect. CONCLUSION: These peptides could reduce the vascular resistance and be used as functional ingredients in the prevention and/or treatment of hypertension and other associated disorders. Copyright © 2010 Society of Chemical Industry     

Comparison of the antioxidant and pro‐oxidant activities of broccoli amino acids with those of common food additives

The antioxidant and pro‐oxidant activities of broccoli amino acids were compared with those of common food additives. In decreasing order, the data showed that Asp, SMC, GABA, Glu, Gln, Pro, Phe, Leu, Lys, Arg, Asn, Val, Ile, His, Ser, Gly, Orn and Ala, when dissolved in water at concentrations of 0.5 and 0.05 mM , partially inhibited damage to deoxyribose in the presence of ferric‐EDTA and H₂O₂. In contrast, Tyr and Thr acted as pro‐oxidants in this system. The amino acids present in broccoli had no hydrogen peroxide‐scavenging effect. When dissolved in water, methanol or ethanol, SMC, Glu, Thr, Gln, Ser, GABA, Pro, Ala, Ile, Phe, Asp, Orn and Tyr inhibited lipid peroxidation. However, Asn, Val, Arg, Leu, Lys, His and Gly were not effective in decreasing peroxidation at concentrations of 0.5 and 0.05 mM . Asp > SMC > Ala > Phe > Hys > Orn > Gln = Ser > Lys > Leu = GABA = Gly > Tyr > Arg = Thr > Val > Asn > Pro > Ile > Glu (p < 0.025) showed scavenging activity towards hypochlorous acid, protecting α₁‐antiproteinase against inactivation. In this paper it has been established that some amino acids premixed with propyl gallate increase its hypochlorous acid‐scavenging capacity, while other amino acids have an additive effect with propyl gallate, permitting smaller quantities of propyl gallate to be used as food additives in some products which contain these amino acids. © 2001 Society of Chemical Industry     

Inhibition properties of dipeptides from salmon muscle hydrolysate on angiotensin I-converting enzyme

We isolated Phe–Leu as an angiotensin I‐converting enzyme (ACE) inhibitor from hydrolysate of chum salmon muscle. The IC₅₀ value of this peptide was 13.6 μm , and it showed non‐competitive inhibition. The reverse sequence dipeptide Leu–Phe also showed ACE inhibitory activity. However, Leu–Phe is much less inhibitory than Phe–Leu with an IC₅₀ value of 383.2 μm . In addition, the inhibition mode was competitive. To investigate the relationship between dipeptide sequence and ACE inhibition properties, we further measured ACE inhibitory activity and inhibition mechanism using six Trp‐containing dipeptides, which had been identified from the same salmon muscle hydrolysate as ACE inhibitory peptides in a previous study. Peptides with Trp as the C‐terminal residue, Ala–Trp, Val–Trp, Met–Trp, Ile–Trp, Leu–Trp showed non‐competitive inhibition. On the other hand, reversed sequence peptides with Trp at the N‐terminal were competitive inhibitors, except Trp–Leu. These results indicate that the sequence of ACE inhibitory dipeptides can affect both inhibitory potency and the inhibition mechanisms.     

Comparative study of partial amino acid sequences of prolamins and glutelins from various cereals. VII. Amino acid sequences of prolamin peptides

The peptide fractions isolated from chymotryptic hydrolysates of wheat, rye and barley prolamines [this journal (1984) 178:173] were separated into pure peptides by high-performance liquid chromatography on octadecyl silica gel. The peptides which were most abundant were analyzed for amino acid composition and in part for amino acid sequence. Besides peptides which are typical for only one of the cereals investigated, peptides of similar composition were found in all three prolamines. These contain repeating sequences and are built up of mainly Gln (Q), Pro (P) and hydrophobic amino acids (X) such as Phe, Tyr, Ile, Val and Leu. One of the most frequent partial sequences is QQPQQPXP.     

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

Egg White Ovotransferrin‐Derived ACE Inhibitory Peptide Ameliorates Angiotensin II‐Stimulated Insulin Resistance in Skeletal Muscle Cells

1 Scope

The renin‐angiotensin system (RAS) is a major contributor to the development of insulin resistance and its related complications. Egg white ovotransferrin‐derived tripeptides, IRW (Ile‐Arg‐Trp), IQW (Ile‐Gln‐Trp), or LKP (Leu‐Lys‐Pro) are previously identified as the inhibitors of angiotensin‐converting enzyme (ACE), a key enzyme in the RAS. This study aims at determining whether these peptides are effective in improving insulin resistance, and their mechanisms of action, in a rat derived skeletal muscle cell line (L6 cells).

2 Methods and results

Insulin resistance is induced by treating L6 cells with 1 μm angiotensin II (Ang II) for 24 h. Effects of peptides on glucose uptake are determined using glucose uptake assay, glucose transporter 4 (GLUT4) translocation by immunofluorescence, reactive oxygen species (ROS) by dihydroethidium (DHE) staining, while insulin signaling pathway, Ang II receptor (AT1R or AT2R) levels, and NADPH oxidase activation are measured using Western Blot. Only IRW treatment significantly improves insulin resistance in L6 cells via stimulation of insulin signaling. IRW decreases Ang II‐stimulated AT1R expression, ROS formation, and NADPH oxidase activation.

3 Conclusions

Of three ACE inhibitory peptides studied, only IRW improves insulin resistance in L6 cells, at least partially via reduced AT1R expression and its anti‐oxidative activity.     

Short communication: Amino acids antagonistic to the amino acids inhibitory for growth rate of mixed ruminal bacteria

Antagonism of some amino acids (AA) to the inhibitory effects of other AA (Ile, Phe, and Thr) on the growth rate of mixed ruminal bacteria was investigated. In vitro growth rate of the mixed ruminal bacteria was inhibited when the 3 inhibitory AA (1 mM each) were each added to individual control treatments in which an ammonium salt was included as a sole N source. The inhibitory effect caused by Ile was relieved by addition of Leu or Val (equimolar to Ile), and no significant inhibition was shown when both Leu and Val were added together with Ile. The growth inhibition caused by Phe was also alleviated by supplementing with Trp, and was completely negated by adding Tyr. The inhibitory effect of Thr, on the other hand, was not affected by addition of Lys or Met (which are synthesized using a common pathway with Thr), but was mitigated by supplementation with Glu, Ser, Val, Ala, or Gln. Among the antagonistic AA, Leu, Val, Trp, Tyr, and Glu were indispensable for the maximum growth rate of the ruminal bacteria under the experimental condition of supplementation of amino-N, the removal of which from a mixture of 20 protein AA caused the growth rate to decline. Removals of Ile along with Leu or Val or both, of Phe along with Trp or Tyr, and of Thr along with Glu recovered the promotion of bacterial growth rate. It was concluded that inhibitions of the bacterial growth rate caused by Ile, Phe, or Thr could be antagonized by some other AA (Leu, Val, Tyr, Trp, or Glu), and the role of these latter AA as relievers of the inhibitory effects could explain why they are indispensable for maximum growth rate of ruminal bacteria.     

Molecular characterization of peptides released from beta-lactoglobulin and alpha-lactalbumin via cardosins A and B

Crude mixtures of aspartic proteases from flowers of the plant Cynara cardunculus have been studied frequently, as have activities of such enzymes (in pure form) on caseins from bovine, ovine, and caprine sources. This research study addressed pure bovine whey protein as substrates; that is, α-lactalbumin (αLA) and β-lactoglobulin (α-LG), submitted to hydrolysis by 1 of 2 aspartic proteases (cardosins A and B), previously extracted and purified from C. cardunculus. Samples collected, following incubation at 55°C and pH 5.2, were assayed by fast protein liquid chromatography, reversed phase-high performance liquid chromatography, and tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis; the major peptides released were then collected and sequenced by Edman degradation. Cardosin B and, to a lesser degree, cardosin A showed proteolytic activity toward α-LA, but the hydrolyzates produced were characterized by distinct peptide profiles. Cardosin B possesses a broad specificity, and produces several hydrophobic peptides (at least 5, with molecular mass in the range 2 to 8 kDa) in the early stages, which eventually become more hydrophilic (with molecular mass below 2 kDa) at later stages of hydrolysis. Cardosin A was found to cleave α-LA at the peptide bonds Phe28-Arg29, Gly54-Tyr55, Ala59-Ile60, Leu71-Phe72, and Leu105-Thr106, whereas cardosin B cleaved Ala19-Glu20, Phe28-Arg29, Glu30-Leu31, Tyr37-Gly38, Trp45-Val46, Phe50-His51, Ala59-Ile60, Ser66-Thr67, Leu71-Phe72, Phe72-Gln73, Gln73-Ile74, Ile78-Trp79, Leu115-Asp116, and Leu124-Ala125. Conversely, cardosins A and B are apparently not active on β-LG.     

Kinetics of amino acid extraction by lactating mammary glands in control and sometribove-treated Holstein cows.

Studies of mammary arteriovenous difference were conducted on multiparous lactating Holstein cows (n = 21) on d 35, 70, 105, and 126 of lactation to examine kinetic relationships between arterial concentration and mammary gland extraction of AA. Additionally, these cows were paired by previous lactational performance and assigned to bST-treated or control groups to examine the effect of bST treatment on AA concentration and extraction by lactating mammary glands. Treated cows were injected daily with 40 mg of recombinant bST from d 71 through 126 of lactation. Arterial concentrations of Asp, Ser, Asn, Gly, beta-aminoisobutyrate, and Met were increased. Concentrations of Val, Ile, Leu, Phe, Orn, and Lys were decreased in bST-treated cows compared with controls. Increased extractions of Asp and Met by mammary glands in treated versus control cows were correlated positively with treatment-induced changes in arterial concentrations of these AA. However, increased mammary extractions of Arg, cystathionine, Leu, and Lys by bST-treated compared with control cows were not correlated with bST-induced changes in arterial concentrations of these AA. Extractions of Asn, His, Thr, Arg, Tyr, Met, cystathionine, cystine, Ile, Phe, Orn, Glu, Gly, Tau, Cit, Leu, and Val were correlated linearly with arterial concentrations (r2 greater than .15) of each AA. Extractions of Asp, Glu, Ser, Asn, Gly, Gln, Tau, His, Cit, Thr, Pro, Tyr, Val, cystine, Ile, Leu, Trp, Orn, and Lys also were correlated with arteriovenous differences of Met.     

SUBSTRATE SPECIFICITY OF AMINOPEPTIDASE FROM JAPANESE CLASSIFIED BARLEY FLOUR

In this study, some properties of a peptidase obtained from Japanese barley were investigated. Boc‐Val‐Leu‐Lys‐MCA was slightly hydrolyzed, but the enzyme showed almost no activity on Suc‐Ala‐Ala‐Pro‐Phe‐MCA and Ac‐Val‐Glu‐Ile‐Asp‐MCA. The enzyme activity decreased to 57.7% by the addition of 0.235 mM bestatin. All tripeptides and tetrapeptides studied were cleaved from the N‐terminus amino acid by the enzyme. However, the enzyme did not cleave Leu‐Pro‐Phe‐Phe‐Asp in the manner of the AP type. The C‐terminus amino acid residue (Asp) and the second amino acid residue of the C‐terminus (Phe) were released at almost the same time. From these findings, this enzyme was identified as both an AP and oligopeptidase. This property is very similar to that of cathepsin H and the novel peptidase purified from mesquite pollen. This enzyme may not only supply useful foodstuffs such as a meat tenderizer but also produce bioactive peptides.     

Isolation of Peptides with Angiotensin I-converting Enzyme Inhibitory Effect Derived from Hydrolysate of Upstream Chum Salmon Muscle

In order to utilize upstream chum salmon as a component of nutraceutical food, their defatted muscle proteins were hydrolyzed with 5% thermolysin. The resulting hydrolysate showed high inhibitory activity against angiotensin I‐converting enzyme (inhibitory concentration₅₀= 27.9 protein μg/mL) in vitro. A significant reduction of systolic blood pressure was observed when 500 and 2000 mg/kg of body weight were orally administered into spontaneously hypertensive rats. Angiotensin I‐converting enzyme inhibitory peptides contained in the hydrolysate were isolated with various chromatographs. These 6 active peptides were Trp residue‐containing dipeptides: Trp‐Ala, Val‐Trp, Trp‐Met, Met‐Trp, Ile‐Trp, and Leu‐Trp. The inhibitory concentration₅₀ values of these dipeptides ranged from 2.5 μM to 277.3 μM.     

Isolation and characterization of angiotensin I‐converting enzyme inhibitory peptides from wheat gliadin hydrolysate

Angiotensin I‐converting enzyme (ACE) inhibitory peptide was isolated from wheat gliadin hydrolysate prepared with acid protease. Consecutive purification methods were used for peptide isolation including ion‐exchange chromatography, size‐exclusion chromatography, and reverse‐phase high‐performance liquid chromatography. The amino acid sequence of this peptide was identified as Ile‐Ala‐Pro, and the ACE inhibitory activity (IC₅₀ value) was 2.7 μM . The hypotensive activity of Ile‐Ala‐Pro on spontaneously hypertensive rats was investigated. This peptide inhibited the hypertensive activity of angiotensin I with intravenous injection, and decreased the blood pressure significantly with intraperitoneal administration.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

Two Novel Bioactive Peptides from Antarctic Krill with Dual Angiotensin Converting Enzyme and Dipeptidyl Peptidase IV Inhibitory Activities

Inhibition of dipeptidyl peptidase IV (DPP‐IV) and angiotensin converting enzyme (ACE) are considered useful in managing 2 often associated conditions: diabetes and hypertension. In this study, corolase PP was used to hydrolyze Antarctic krill protein. The hydrolysate (AKH) was isolated by ultrafiltration and purified by size‐exclusion chromatography, ion exchange chromatography and reversed‐phase high‐performance liquid chromatography (RP‐HPLC) sequentially. The in vitro inhibitory activities of all AKHs and several fractions obtained against ACE and DPP‐IV were assessed. Two peptides, purified with dual‐strength inhibitory activity against ACE and DPP‐IV, were identified by TOF‐MS/MS. Results indicated that not all fractions exhibited dual inhibitory activities of ACE and DPP‐IV. The purified peptide Lys‐Val‐Glu‐Pro‐Leu‐Pro had half‐maximal inhibitory concentrations (IC₅₀) of 0.93±0.05 and 0.73±0.04 mg/mL against ACE and DPP‐IV, respectively. The other peptide Pro‐Ala‐Leu had IC₅₀ values of 0.64±0.05 and 0.88±0.03 mg/mL against ACE and DPP‐IV, respectively. This study firstly reported the sequences of dual bioactive peptides from Antarctic krill proteins, further provided new insights into the bioactive peptides responsible for the ACE and DPP‐IV inhibitory activities from the Antarctic krill protein hydrolysate to manage hypertension and diabetes.     

Distribution of stable free radicals among amino acids of isolated soy proteins

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.     

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

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

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Free amino acid composition in primary and secondary inflorescences of 11 broccoli (Brassica oleracea var italica) cultivars and its variation between growing seasons

Eleven broccoli cultivars were grown in the field in spring/summer (April–July) and summer/winter (September–January). Free amino acid composition was determined by HPLC in primary and secondary inflorescences separately. A total of 17 amino acids were identified: L ‐alanine (Ala), L ‐arginine (Arg), L ‐asparagine (Asn), L ‐aspartic acid (Asp), glycine (Gly), L ‐glutamic acid (Glu), L ‐glutamine (Gln), L ‐histidine (His), L ‐isoleucine (Ile), L ‐leucine (Leu), L ‐methionine (Met), L ‐phenylalanine (Phe), L ‐serine (Ser), L ‐threonine (Thr), L ‐tryptophan (Trp), L ‐tyrosine (Tyr) and L ‐valine (Val). The major amino acid was L ‐glutamine, which represented on average between 39.5% (in cvs Durango and Green Valiant) and 55.5% (in cv Shogun) of the total amino acid content among cultivars, followed by L ‐glutamic acid with a variation between 12.1% (in cv Shogun) and 17.4% (in cv Marathon). A few amino acids represented less than 1% each (Gly, Leu, Met, Phen, Thr, Trp and Tyr). For most of the amino acids there were significant differences between cultivars, whilst only a few amino acids showed significant variations between inflorescences. Season also induced significant differences in the content of most of the identified amino acids. The cultivar with the highest total free amino acid content (323.9 mmol kg⁻¹ DW) on average of both seasons (391.3 in spring/summer and 256.4 in summer/winter) was Shogun, whilst the others were above the minimum of 177.3 mmol kg⁻¹ DW found in cv SK3. There was a general tendency for higher total amino acids levels in spring/summer than in summer/winter, but it was clear that this effect was dependent on the cultivar. © 2000 Society of Chemical Industry     

Angiotensin I‐converting enzyme inhibitory peptides FQPSF and LKYPI identified in Bacillus subtilis A26 hydrolysate of thornback ray muscle

Angiotensin I‐converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC₅₀ of 0.83 mg mL^?1. To identify peptides responsible for this activity, the extract was eluted through size‐exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC₅₀ of 0.42 and 0.51 mg mL^?1, respectively. These fractions were analysed by nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC₅₀ of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides.