Optimising the free radical scavenging activity of shrimp protein hydrolysate produced with alcalase using response surface methodology

Response surface methodology (RSM) was used to optimise the hydrolysis parameters of Acetes chinensis by Alcalase 2.4L in order to obtain a hydrolysate with potent radical scavenging activity. The parameters were temperature, pH and enzyme concentration/substrate concentration (E/S) ratio with degree of hydrolysis (DH) being the response. The results showed that the optimum condition was: temperature at 57 °C, pH at 8.0, E/S ratio at 2.6AU 100 g⁻¹ shrimp, hydrolysis time 3 h. The DH was 26.32%, the hydroxyl radical scavenging activity was up to 88.12% and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was 35.61%. The gel column filtration chromatography by a Sephadex G-25 column yielded five fractions. The molecular weight of the most potent free radical scavenging activity fraction ranged from 915 to 207 Da, its IC₅₀ for hydroxyl radical was 0.03 mg mL⁻¹, and IC₅₀ for DPPH radical was 8.86 mg mL⁻¹.     

Identification of an Angiotensin I-converting Enzyme Inhibitory Peptides from Protein Hydrolysates by a Soybean Protease and the Antihypertensive Effects of Hydrolysates in 4 Spontaneously Hypertensive Model Rats

ABSTRACT: Our previous study demonstrated that, because of its substrate specificity, protein hydrolysates by protease D3, which is originated from soybean, exhibited the prominent property of being less bitter than other enzymatic hydrolysates. In the 1st experiment in this series, angiotensin I-converting enzyme (ACE) inhibitory peptides from soy protein hydrolysate by D3 were identified by the establishment of a novel and effective peptide identification method. The amino acid sequences of candidate ACE inhibitory peptides were determined by electrospray ionization mass/mass spectrometry (MS/MS) analysis after rough purification of the samples with gel filtration chromatography and reverse-phase chromatography. Some of the candidate peptides had amino acid sequences that showed homology with those of the reported ACE inhibitory peptides. Then, 8 types of novel candidate peptides were synthesized according to a solid-phase method, and their ACE inhibitory activity was confirmed as the IC₅₀ value. The most potent inhibitor was NWGPLV (IC₅₀= 21 μ M ). In the 2nd experiment, the antihypertensive activity of protein hydrolysates by D3 was investigated in spontaneously hypertensive model rats (SHRs). The dose-dependent antihypertensive effect of soy protein hydrolysate was confirmed, and systolic blood pressure was significantly reduced after the oral administration of doses exceeding 100 mg/kg. Casein hydrolysate was found to have the most potent effects on suppressing blood pressure as well as ACE inhibitory activity among the various food protein hydrolysates studied because of the primary structure of casein. These results indicate that hydrolysates by D3 could be a useful food ingredient because it has the physiological function (antihypertensive activity).     

Vascular effects and antihypertensive properties of κ-casein macropeptide

The blood-pressure-lowering effect of bovine κ-casein macropeptide (CMP), previously reported to exhibit a modest angiotensin-converting enzyme (ACE)-inhibitory activity in vitro, was evaluated in spontaneously hypertensive rats (SHR). The oral administration of CMP (150 mg kg⁻¹) significantly reduced the systolic blood pressure (SBP) of SHR. The antihypertensive action was more pronounced when CMP was treated with trypsin. The sequence MAIPPKK, identified in the tryptic hydrolysate of bovine CMP, significantly reduced blood pressure at a dose of 10 mg kg⁻¹. The CMP and its tryptic peptides induced relaxation of endothelium-intact aortic rings. The sequence MAIPPKK also evoked a significant relaxation effect; however, the shorter sequence MAIPPK and the strong ACE-inhibitory tripeptide IPP exhibited a vascular relaxing effect lower than 10%. The implications of vascular relaxing mechanisms, as well as the possibility that the tripeptide IPP is at least partially responsible for the antihypertensive effects of CMP, are discussed.     

Purification and identification of an ACE inhibitory peptide from the peptic hydrolysate of Acetes chinensis and its antihypertensive effects in spontaneously hypertensive rats

Acetes chinensis is a marine shrimp found in the coastal waters of China. The shrimp was hydrolysed by pepsin to prepare hydrolysates with angiotensin I‐converting enzyme (ACE) inhibitory activity. The hydrolysate with the highest ACE inhibitory activity resulted from a 3–5 h incubation at 45 °C and pH 2.5 with pepsin. Gel filtration and RP‐HPLC were used to separate ACE inhibitory peptides from the hydrolysate. The gel filtration fraction of the hydrolysate with a molecular weight range from 1320 Da to 311 Da exerted the highest ACE inhibition activity. This fraction was separated by RP‐HPLC into fifteen fractions, of which fraction F9 showed 92.7% of the ACE inhibition activity. Its peptide sequence was determined to be Leu–His–Pro. It showed a potent antihypertensive activity in spontaneously hypertensive rats. The results suggested that this peptide may be a potent ACE inhibitor which might be developed into a healthy food to lower blood pressure.     

SOYBEAN PROTEIN-DERIVED HYDROLYSATE AFFECTS BLOOD PRESSURE IN SPONTANEOUSLY HYPERTENSIVE RATS

The aim of this study was to investigate the inhibitory activity of angiotensin-converting enzyme (ACE) and antihypertensive effects of soybean protein hydrolysate in spontaneously hypertensive rats (SHRs). The inhibitory activities against ACE with an IC₅₀ of the peptidic fraction were 0.82 (casein), 0.73 (soybean protein isolate), and 0.48 mg/mL (soybean acid-precipitated protein), respectively. Peptic hydrolysate containing 1% NaCl was added to the SHRs'feed (5% of each protein hydrolysate). Systolic blood pressure of the soybean protein hydrolysate-supplemented groups were significantly lower than that of the casein hydrolysate-supplemented group in the study period. These data suggested that soybean protein hydrolysate may retard the development of hypertension in SHRs by its ACE inhibitory effect in vivo.     

LC-MS method development to evaluate major triterpenes in skins and cuticular waxes of grape berries

Predominant triterpenes (free oleanolic acid, β-sitosterol and β-sitosterol-3-O-β- d -glucoside) in skin and cuticular wax of grape berries have been analysed by liquid chromatography–mass spectrometry (LC-APCI-MS). The validity of the developed method was established by determining linearity, recovery, precision, accuracy, limit of detection and quantification. Detection limits were in the range of 0.12–0.25 mg L⁻¹, and linearity values ranged up to 105 mg L⁻¹. The repeatability of the method was good. High variability was found among the measured grape varieties based on triterpene content, quantitites of oleanolic acid of cuticular wax ranged from 31.53 mg kg⁻¹ to 162.01 mg kg⁻¹ of the twelve analysed samples. The highest sitosterol content was measured in the sample 'Othello' (73.12 mg kg⁻¹), while maximum sitosterol glucoside content was also found in 'Othello' variety (13.68 mg kg⁻¹). The results showed that the study on triterpenes could be an informative tool to characterise grape varieties.     

Solubilization of onion with polysaccharide-degrading enzymes

For the selection of suitable enzymes for the solubilization of onion, degree of solubilization (DS) values were measured. The DS values of Pectinex and Viscozyme were 75.8 and 78.4%, respectively, which indicates they have higher specific activities than Cereflo and Celluclast. The enzyme mixture of Pectinex and Viscozyme (relative ratio of 1:3) had higher DS values and reducing sugar content than Pectinex alone. The enzyme mixture degraded onions with a synergistic effect, solubilizing 85% of the onion. The DS values and reducing sugar content at the optimal condition (pH 4.5 and 45 °C) reached a maximum of 85% and 494.8 mg g^–1 of onion, respectively. The DS values and reducing sugar content increased with increasing reaction time, reaching a maximum of 89% and 517.5 mg g^–1 of onion, respectively. When cooking pork, onion appeared to be preferable to onion hydrolysate, however there was no significant difference. The sweetness and preference of pork cooked with 3% addition of hydrolysate per gram of pork meat were the highest but those were not different significantly from those cooked with less than 10% addition of hydrolysate per pork meat.     

Radical scavenging and antimicrobial activity of horsetail (Equisetum arvense L.) extracts

A reversed-phase high performance liquid chromatography (RP-HPLC) separation on C₈ column and quantitative method were developed to analyse hydroxyl derivatives of benzoic and cinnamic acid and flavonoids in horsetail ( Equisetum arvense L.) extracts. Total phenolic content of n -butanol, ethyl acetate and water extracts, determined by the Folin-Ciocalteu method, was 96.4, 26.4 and 15.4 mg g⁻¹ of dry extracts, respectively. The antioxidative activity of horsetail extracts was tested by measuring their ability to scavenge stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reactive hydroxyl radicals by electron spin resonance spectroscopy. The results demonstrated that the free radical scavenging activity (versus both DPPH and hydroxyl radicals) depended on the type and concentration of applied extracts; the highest DPPH (EC₅₀ = 0.65 mg mL⁻¹) and hydroxyl radical scavenging activities (EC₅₀ = 0.74 mg mL⁻¹) were obtained in the case of n -butanol extract. The radical scavenging activity of extracts significantly correlated with total phenolic content. The antimicrobial tests showed that ethyl acetate and n -butanol extracts inhibited the growth of tested bacteria.     

Natural preservative ingredient from marine alga Ulva lactuca L.

The contents of total chlorophyll (T-Chl), carotenoids and phenolics compounds were quantified in the biomasses of Ulva lactuca grown either in normal or artificial sea water under indoor conditions. The antioxidant and antibacterial activities of U. lactuca crude organic extracts ( Ulva- COEs) were determined. Thirty-four compounds in Ulva- COEs were characterised by thin layer chromatography and high-performance liquid chromatography. The major compounds were chlorophyll a (Chl a ) (15.60–30.90%) and b (Chl b ) (12.20–14.89%) , 9-cis β-carotene (13.12–14.47%), α-carotene (11.44–11.47%) and all-trans β-carotene (6.16–29.70%, of total carotenoids).The Ulva- COEs exhibited remarkable antioxidant activity, with an IC₅₀ (concentration which causes a 50% of DPPH radical scavenging activity) values ranged from 16.5 and 18.7 μg mL⁻¹, which could be compared with the synthetic antioxidants: α-tocopherol (14.4 μg mL⁻¹), butylated hydroxyanisol (13.1 μg mL⁻¹) and butylated hydroxyltoluene (13.1 μg mL⁻¹). Also, Ulva- COEs exhibited great potential antibacterial activities against six bacterial strains, with minimal inhibitory concentration values ranged from 0.40 to 0.35 mg mL⁻¹.     

Antioxidants in aerial parts of Hypericum sampsonii, Hypericum japonicum and Hypericum perforatum

Antioxidants contents and antioxidative enzymes and their activities in fresh aerial tissues of Hypericum sampsonii (Sampson's St John's Wort), Hypericum japonicum (Japanese St John's Wort) and Hypericum perforatum were investigated. Hypericum sampsonii contained more total ascorbate [34.33 μmol g⁻¹ fresh weight (FW)] than H. perforatum (57% less) and H. japonicum (82% less). It also contained more thiol and phenolics than two other species. Hypericum japonicum had highest superoxide dismutase (SOD) activity (8.74 mmol min⁻¹ g⁻¹ FW), followed by H. sampsonii (2% less) and H. perforatum (37% less). Hot-air dried H. perforatum materials contained more thiol [208.7 μmol g⁻¹ dry weight (DW)] and phenolics (352.82 mg g⁻¹ DW) than freeze-dried and fresh materials. Both drying treatments decreased the activities of antioxidative enzymes in aerial tissues of H. perforatum . However, freeze-dried H. perforatum contained the highest SOD activity (5.42 mmol min⁻¹ g⁻¹ DW) among the antioxidative enzymes measured from both freeze-dried and hot-air dried tissues (ranged from 0.02 to 2.65 μmol min⁻¹ g⁻¹ DW).