Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

Breaking the spores of the fungus Ganoderma lucidum by supercritical CO2

The hard sporoderm of Ganoderma lucidum spores prevents the release of bioactive components such as polysaccharides which have significant anti-tumour activity. In the present study, supercritical carbon dioxide (SC–CO₂) was used for the sporoderm breaking of G. lucidum spores, and polysaccharides were subsequently extracted and determined for evaluating the performances of SC–CO₂. The operating parameters were optimized by orthogonal array design (OAD), and the morphological status of sporoderm was observed by scanning electron microscope (SEM). The optimum operating conditions for SC–CO₂ breaking of sporoderm were as follows: pressure 35 MPa, temperature 25 °C, time 4 h, and CO₂ flow rate 10 kg/h. After SC–CO₂ processing, the extraction yield of polysaccharides reached 2.98%, which was 3-fold to that of the intact ones (0.94%). This method is fast, efficient and advanced enough to break the hard sporoderm of G. lucidum, which may provide a scientific reference for the large-scale processing of spores in the pharmaceutical and food industries.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

Solid–liquid expression from denaturated plant tissue: Filtration–consolidation behaviour

The objective of this study is to investigate the filtration–consolidation behaviour of biological tissue (sugar beet specimens), denaturated by pulsed electric field (PEF) and freezing–thawing pretreatments. The degree of cell membrane damage was evaluated by the tissue electrical conductivity measurement (determination of the cell disintegration index Z). The PEF-treated and freeze–thawed tissues show different structure response on the applied compressive pressure and different filtration–consolidation behaviour. The value of fracture pressure (P_f ≈ 58.5 bar) is the lowest and the value of tissue deformation under fracture (ε_f ≈ 0.46) is the highest for sugar beet tissue treated by freezing/thawing. PEF treatment leads to more rigid and more fragile tissue structure (P_f ≈ 63.8 bar, ε_f ≈ 0.28) than freeze–thawed tissue. Untreated tissue seems to be stronger and less fragile than the PEF-treated tissue (P_f ≈ 84.1 bar, ε_f ≈ 0.37). Different tissue reactions on the loading pressure evidently reflect the difference between microstructures of treated and untreated tissues. The filtration/consolidation behaviour during liquid expression from a sugar beet tissue also depends on the type and degree of structure denaturation. Tissues treated by PEF and freezing/thawing demonstrate two consolidation stages of expression (primary and secondary). However, the freeze–thawed tissue tends to modify the expression mechanism under the higher loading pressure (60 bar) with approach to the primary consolidation behaviour. The simplified semi-empirical consolidation model permits a reasonably good prediction of expression behaviour from the denaturated sugar beet tissue and estimation of consolidation coefficient b under different pressures. The extrapolation of curves b(P) shows the value of hypothetical pressure, P_max ≈ 90 bar at which the consolidation behaviour would be similar to both PEF-treated and freeze–thawed tissues.     

Antibacterial peptides from barbel muscle protein hydrolysates: Activity against some pathogenic bacteria

Peptides obtained by enzymatic hydrolysis of fish proteins exhibit not only nutritional but also biological properties of dietary uses, or even therapeutic potential. The objective of the present study was to isolate and characterize peptides from the protein hydrolysates of barbel muscle with antibacterial activity against Gram-positive (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter sp.) bacteria. Barbel muscle protein hydrolysates (BMPHs), obtained by treatment with Alcalase^® (DH = 6.6%), was fractionated by size exclusion chromatography on a Sephadex G-25 and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses and amino acid sequences of these peptides were determined using ESI–MS and ESI–MS/MS, respectively. Eleven peptides in F_II-1, F_II-2, F_II-3 and F_II-4 sub-fractions separated by RP-HPLC were identified. The most active peptide fraction (F_II-3) contained three peptides: Ala–Ala–Ala–Leu; Ala–Ala–Gly–Gly–Val and Ala–Ala–Val–Lys–Met.     

Triglycerides constituted of short and medium chain fatty acids and dicarboxylic acids in Momordica charantia, as well as capric acid,inhibit PGE2 production in RAW264.7 macrophages

This study was aimed to identify compounds in bitter gourd (Momordica charantia) ethyl acetate extract (EAE), which inhibit lipopolysaccharide-induced prostaglandin E₂ (PGE₂) production in RAW264.7 cells. Bitter gourd EAE was partitioned between n-hexane and methanol/H₂O (90/10). The hexane fraction was further separated by repeated silica gel chromatographies, and a reverse phase (RP) C18 chromatography. Fraction RP-10 showed the highest inhibition effect on PGE₂ production (Max inhibition = 96%, IC₅₀ = 2.3 μg/ml) and was identified to be triglycerides constituted of short and medium chain fatty acids by ¹H NMR, IR and H–HCOSY, and dicarboxylic acids by GC/MS. Fatty acids with 3–20 carbons were tested for the inhibitory activity, and capric acid exhibited the highest effect (Max inhibition = 99%, IC₅₀ = 6.5 μM). In conclusion, triglycerides composed of short and medium chain fatty acids and dicarboxylic acids in bitter gourd inhibit PGE₂ production, and capric acid is the most potent inhibitor among the fatty acids.     

Production of dipeptidyl peptidase IV inhibitory peptides from defatted rice bran

The insulinotropic hormone glucagon-like peptide-1 is metabolised extremely rapidly by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, human DPP-IV is a key regulator involved in the prevention and treatment of type 2 diabetes. To simplify the method of producing an inhibitory peptide against DPP-IV, we focused on rice bran (RB) as a source and subjected proteins from defatted RB to enzymatic proteolysis using 2 commercial enzymes. The RB peptides produced with Umamizyme G exhibited 10 times the inhibitory activity as those produced with Bioprase SP. The half-maximal inhibitory concentration (IC₅₀) value of the RB peptides was 2.3 ± 0.1 mg/ml. Leu-Pro and Ile-Pro were identified as the inhibitory peptides among the RB peptides produced with Umamizyme G. Ile-Pro was the strongest DPP-IV inhibitor among the 15 Xaa-Pro dipeptides and Pro-Ile tested. Ile-Pro competitively inhibited DPP-IV (K_i = 0.11 mM). Mass spectrometry indicated that the contents of Leu-Pro and Ile-Pro in the RB peptides were 2.91 ± 0.52 μg/mg.     

Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product

To produce and identify bioactive peptides, two commercial enzymes, orientase (OR) and protease XXIII (PR) were used to hydrolyze tuna dark muscle by-product for up to 6 h, and the hydrolysates were evaluated for antioxidative properties. The results showed that, 60-min OR and 120-min PR hydrolysates possessed the highest antioxidative activity. Then, the protein hydrolysates were subjected to a Sephadex G-25 gel filtration chromatography, and the molecular weight of the peptide fractions which showed the highest antioxidative activity ranged from 390 to 1400 Da. The peptide fractions were further isolated using the two-step high-performance liquid chromatography (HPLC-1 and HPLC-2), and the amino acid sequences of the two antioxidative peptides from OR and PR hydrolysates were Leu–Pro–Thr–Ser–Glu–Ala–Ala–Lys–Tyr (978 Da) and Pro–Met–Asp–Tyr–Met–Val–Thr (756 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna dark muscle by-product may be useful ingredients in food and nutraceutical applications.     

Angiotensin I-converting enzyme-inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain

A number of biopeptides promoting health benefits have been isolated from food-protein hydrolysates and can be released during enzymatic digestion. Antihypertensive peptides can be part of protein fractions from amaranth grain. The objective of this work was to obtain ACE-inhibitory peptide fractions from albumin 1 and the globulin of amaranth (Amaranthus hypochondriacus) grain. Albumin 1 and globulin were hydrolysed with alcalase; hydrolysis was monitored by proteolytic degradation and by ACE-inhibitory activity. The highest ACE-inhibitory activity was 40% and 35% as obtained after 18 and 15 h hydrolysis for albumin 1 and globulin, respectively. Further separation and purification of the ACE-inhibitory peptide fractions were carried out by gel filtration and C₁₈ RP–HPLC. The IC₅₀ was 0.35 ± 0.02 mg/ml for albumin 1 peptide fraction and 0.15 ± 0.03 mg/ml for globulin peptide fraction. Albumin 1 peptide fraction showed an competitive mode of ACE inhibition, whereas the globulin peptide fraction was competitive. The globulin peptide fraction may have one of the most active naturally-occurring ACE-inhibitory peptides.     

Predictive modelling of angiotensin converting enzyme inhibitory dipeptides

The ability of docking to predict angiotensin converting enzyme (ACE) inhibitory dipeptide sequences was assessed using AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Peptide intestinal stability was assessed using a prediction amino acid clustering model. Selected dipeptides, having AutoDock Vina scores ? −8.1 and predicted to be ‘stable’ intestinally, were characterised, using LIGPLOT and for ACE-inhibitory potency. Two newly identified ACE-inhibitory dipeptides, Asp-Trp and Trp-Pro, having Vina scores of −8.3 and −8.6 gave IC₅₀ values of 258 ± 4.23 and 217 ± 15.7 μM, respectively. LIGPLOT analysis indicated no zinc interaction for these dipeptides. Phospho-dipeptides were predicted to have a good affinity for ACE. However, the experimentally determined IC₅₀ results did not correlate since, for example, Trp-pThr and Pro-pTyr, having Vina scores of −8.5 and −8.1, respectively, displayed IC₅₀ values of >500 μM. While docking allowed identification of new ACE inhibitory dipeptides, it may not be a fully reliable predictive tool in all cases.     

A proposed mechanism of tenderising post-rigor beef using high pressure–heat treatment

Tenderness of beef M. Sternomandibularis was tough when cooked from both raw, and when previously heated (60 °C, 20 min), whereas a significant improvement in tenderness was achieved when pressure–heat (P–H) treated muscle (200 MPa, 60 °C, 20 min) was cooked. In order to determine the mechanism for this improvement, connective tissue, myofibrillar and sarcoplasmic proteins, were separated into three fractions and studied with regard to their solubilisation, denaturation and aggregation, degradation and strengthening of protein structures for the three treatments (raw, heated and H–P treated). Measurements included DSC, SDS–PAGE, surface hydrophobicity, and the appearance, length and width of myofibres (light microscopy). For the connective tissue fraction, heat solubility was determined.     

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31: (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl₂; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).     

The influence of latitude on the concentration of vitamin D3 and 25-hydroxy-vitamin D3 in Australian red meat

There is little information on the vitamin D content of Australian red meat or on the possible influence of latitude on this content. To determine the content of vitamin D₃ and 25-hydroxy-vitamin D₃ (25OHD₃), lamb and beef were analysed from 34° S with LC–IT-MS. To investigate the possible influence of latitude on vitamin D in meat, the lean meat and fat from five cuts of beef were analysed from 17° S and 41° S. Lamb contained 0.10 μg vitamin D₃/100 g and 0.20 μg 25OHD₃/100 g lean meat, while beef contained 0.12 μg vitamin D₃ and 0.27 μg 25OHD₃/100 g (lean meat). Latitude had no effect on the vitamin D₃ (P = 0.21) or 25OHD₃ (P = 0.29) content of lean beef, but fat from cattle in the 17° S latitude group contained significantly higher (P < 0.01) concentrations of vitamin D₃ than fat from the 41° S group of cattle.     

Effect of ohmic heating and vacuum impregnation on the quality and microbial stability of osmotically dehydrated strawberries (cv. Camarosa)

The combined effect of osmotic dehydration/ohmic heating (OD–OH) and vacuum impregnation/ohmic heating (VI–OH) on physicochemical and quality parameters of strawberry (a_w, color, firmness and microstructure), as well as on microbial stability of storage samples at 5 and 10 °C, was analyzed. Treatments were carried out with a 65% (w/w) sucrose solution at 30 °C, and ohmic heating at 9.2, 13, and 17 V/cm electric field strengths, corresponding to applied voltages of 70, 100, and 130 V. Dehydrated samples showed that water loss was greater in OD–OH treatments at 17 V/cm. The greatest solute gain, least firmness loss and least color loss were obtained in the VI–OH treatment at 13 V/cm. The shelf-life of strawberries treated with VI–OH at 13 V/cm and stored at 5 °C was extended from 12 d (control samples) to 25 d. Furthermore, the VI–OH treatment at 13 V/cm was the best processing condition for dehydrating strawberries.     

Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pretreatment and extraction time

Gelatins from the skin of unicorn leatherjacket (Aluterus monoceros) pretreated with different acids (0.2 M acetic acid or 0.2 M phosphoric acid) and extracted with distilled water at 45 °C for various times (4 and 8 h) were characterized. Yields of 5.23–9.18 or 6.12–11.54% (wet weight basis) were obtained for gelatins extracted from the skin pretreated with 0.2 M acetic acid or 0.2 M phosphoric acid, respectively. Extracted gelatins contained α₁ and α₂ chains as the predominant components and some degradation peptides. The absorption bands of gelatins in FTIR spectra were mainly situated in the amide band region (amide I, amide II and amide ???) and showed the significant loss of molecular order of triple helix. Gelatin samples had a relative solubility greater than 90% in the wide pH ranges (1–10). The gel strength of gelatin from skin pretreated with phosphoric acid (GPA) was higher than that of gelatin from skin pretreated with acetic acid (GAA). Both GPA and GAA had the lower gel strength than that of commercial bovine gelatin (P < 0.05). Net charge of GAA and GPA became zero at pHs of 6.64–7.15 and 6.78–7.26, respectively, as determined by zeta potential titration. Emulsifying and foaming properties of GAA and GPA increased with increasing concentrations (1–3%, w/v). Those properties were governed by pretreatments and extraction time. Thus gelatin can be successfully extracted from unicorn leatherjacket skin using the appropriate acid pretreatment and extraction time.     

Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects in vivo

This study aimed to identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from porcine skeletal myosin B. Proteins were hydrolyzed with pepsin and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. The 50% inhibitory concentrations of Lys–Arg–Val–Ile–Gln–Try (M6 a novel peptide) and Val–Lys–Ala–Gly–Phe (A5, a peptide discovered by Ukeda et al. (1991)) were 6.1 and 20.3 μM, respectively. As a result of a homology search, it was determined that the M6 peptide originated from myosin and peptide A5 was of actin origin. M6 is a novel ACE inhibitory peptide, whose activity was shown to be the strongest amongst the previously published myosin-originated peptides. Kinetic evaluations showed that both peptides are competitive inhibitors of ACE. Based on their activity against ACE, M6 was classified as a pro-drug conformer and A5 was classified as a substrate conformer. When both peptides were administered orally to spontaneously hypertensive rats at doses of 10 mg/kg, temporal hypertension was observed after 6 h. This study suggests that M6 and A5 are peptides that may serve several purposes. Based on their remarkable antihypertensive activity, we suggest that M6 and A5 may have potential applications as functional food, which could be used as nutraceutical compounds.     

Multiresidue method for determination of 88 pesticides in berry fruits using solid-phase extraction and gas chromatography–mass spectrometry: Determination of 88 pesticides in berries using SPE and GC–MS

A method using solid phase extraction (SPE) cleanup followed by gas chromatography–mass spectrometry (GC–MS) has been established for quantitative determination of 88 pesticide residues in berry fruits including raspberry, strawberry, blueberry and grape. Based on an appraisal of the characteristics of GC–MS, validation experiments were conducted for 88 pesticides. In the method, solid-phase extraction was carried out using Envi-Carb cartridge coupled with NH₂-LC cartridge with acetonitrile–toluene (3:1, v/v) as the eluted solvent. In the linear range of each pesticide, the correlation coefficient was R² ? 0.99. At the low, medium and high three fortification levels of 0.05–0.5 mg kg⁻¹, recoveries fell within 63–137%. The relative standard deviation was between 1% and 19% for all 88 pesticides. Low limits of detection (0.006–0.05 mg kg⁻¹) and quantification (0.02–0.15 mg kg⁻¹) were readily achieved with this method for all tested pesticides.     

Angiotensin I-converting enzyme inhibitory properties of lentil protein hydrolysates: Determination of the kinetics of inhibition

ACE inhibitory activity was studied for different hydrolysates obtained from protein concentrates of two lentil varieties by in vitro gastrointestinal simulation, Alcalase/Flavourzyme, papain and bromelain. Protein/peptide profiles studied by electrophoresis and HPLC-SEC showed a rich composition of the hydrolysates in small peptides ranging in size from 0.244 to 1.06 kDa. ACE inhibitory activity was measured using the HPLC Hippuryl-His-Leu (HHL) substrate method. Significantly different (P < 0.05) IC₅₀ values ranging between 0.053 and 0.190 mg/ml were obtained for different hydrolysates. Furthermore, the inhibition mechanism investigated using Lineweaver–Burk plots revealed a non-competitive inhibition of ACE with inhibitor constants (K_i) between 0.16 and 0.46 mg/ml. These results demonstrate that hydrolysates of lentil proteins obtained by different enzymatic digestions may contain bioactive components.     

Identification of irradiated meats by determining o- and m-tyrosine as markers

To identify the irradiated meats, various parameters that affect extraction efficiency of tyrosine positional isomers were evaluated. The optimum procedure employed simple extraction by 0.1% formic acid and protein precipitation by acetone. Baseline separation for the extract was carried out on LC–fluorescence detection (FLD) and LC–tandem mass spectrometry (MS/MS). The LC–FLD and LC–MS/MS method had LOD of 1.7–2.1 and 0.3–0.5 ng/mL, respectively, and showed excellent linear correlation over three orders of magnitude, obtained ideal recoveries (78.68–88.90%) and RSD (≤ 8%). The methods were successfully applied in multiple samples. For o- and m-tyrosine, the order of descending trend was: chicken > beef > hairtail > pork and chicken > hairtail > beef > pork, respectively. The radiation dose could be quantitatively evaluated by the nonlinear correlation (y = A₀x² + A₁x + A₂) with coefficients of determination r² > 0.998 in individual meat samples.