Eighteen Novel Bioactive Peptides from Monkfish (Lophius litulon) Swim Bladders: Production, Identification, Antioxidant Activity, and Stability.

In the study, papain was chosen from five proteases to hydrolyze proteins of monkfish swim bladders for effectively utilizing monkfish (Lophius litulon) processing byproducts, and the hydrolysis conditions of papain were optimized as hydrolysis temperature of 65 °C, pH 7.5, enzyme dose 2.5% and time 5 h using single-factor and orthogonal experiments. Eighteen peptides were purified from the swim bladder hydrolysate of monkfish by ultrafiltration and gel permeation chromatography methods and identified as YDYD, QDYD, AGPAS, GPGPHGPSGP, GPK, HRE, GRW, ARW, GPTE, DDGGK, IGPAS, AKPAT, YPAGP, DPT, FPGPT, GPGPT, GPT and DPAGP, respectively. Among eighteen peptides, GRW and ARW showed significant DPPH· scavenging activities with EC50 values of 1.053 ± 0.003 and 0.773 ± 0.003 mg/mL, respectively; YDYD, QDYD, GRW, ARW and YPAGP revealed significantly HO· scavenging activities with EC50 values of 0.150 ± 0.060, 0.177 ± 0.035, 0.201 ± 0.013, 0.183 ± 0.0016 and 0.190 ± 0.010 mg/mL, respectively; YDYD, QDYD, ARW, DDGGK and YPAGP have significantly O2-· scavenging capability with EC50 values of 0.126 ± 0.0005, 0.112 ± 0.0028, 0.127 ± 0.0002, 0.128 ± 0.0018 and 0.107 ± 0.0002 mg/mL, respectively; and YDYD, QDYD and YPAGP showed strong ABTS+· scavenging ability with EC50 values of 3.197 ± 0.036, 2.337 ± 0.016 and 3.839 ± 0.102 mg/mL, respectively. YDYD, ARW and DDGGK displayed the remarkable ability of lipid peroxidation inhibition and Ferric-reducing antioxidant properties. Moreover, YDYD and ARW can protect Plasmid DNA and HepG2 cells against H2O2-induced oxidative stress. Furthermore, eighteen isolated peptides had high stability under temperatures ranging from 25-100 °C; YDYD, QDYD, GRW and ARW were more sensitive to alkali treatment, but DDGGK and YPAGP were more sensitive to acid treatment; and YDYD showed strong stability treated with simulated GI digestion. Therefore, the prepared antioxidant peptides, especially YDYD, QDYD, GRW, ARW, DDGGK and YPAGP from monkfish swim bladders could serve as functional components applied in health-promoting products because of their high-antioxidant functions.

Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from the Collagens of Monkfish (Lophius litulon) Swim Bladders: Isolation, Characterization, Molecular Docking Analysis and Activity Evaluation.

The objective of this study was to isolate and characterize collagen and angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) peptides from the swim bladders of monkfish (Lophius litulon). Therefore, acid-soluble collagen (ASC-M) and pepsin-soluble collagen (PSC-M) with yields of 4.27 ± 0.22% and 9.54 ± 0.51%, respectively, were extracted from monkfish swim bladders using acid and enzyme methods. The ASC-M and PSC-M contained Gly (325.2 and 314.9 residues/1000 residues, respectively) as the major amino acid, but they had low imino acid content (192.5 and 188.6 residues/1000 residues, respectively) in comparison with collagen from calf skins (CSC) (216.6 residues/1000 residues). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and ultraviolet (UV) absorption spectrums of ASC-M and PSC-M illustrated that they were mainly composed of type I collagen. Subsequently, three ACEi peptides were isolated from a PSC-M hydrolysate prepared via a double-enzyme system (alcalase + neutrase) and identified as SEGPK (MHP6), FDGPY (MHP7) and SPGPW (MHP9), with molecular weights of 516.5, 597.6 and 542.6 Da, respectively. SEGPK, FDGPY and SPGPW displayed remarkable anti-ACE activity, with IC50 values of 0.63, 0.94 and 0.71 mg/mL, respectively. Additionally, a molecular docking assay demonstrated that the affinities of SEGPK, FDGPY and SPGPW with ACE were -7.3, -10.9 and -9.4 kcal/mol, respectively. The remarkable ACEi activity of SEGPK, FDGPY and SPGPW was due to their connection with the active pockets and/or sites of ACE via hydrogen bonding, hydrophobic interaction and electrostatic force. Moreover, SEGPK, FDGPY and SPGPW could protect HUVECs by controlling levels of nitric oxide (NO) and endothelin-1 (ET-1). Therefore, this work provides an effective means for the preparation of collagens and novel ACEi peptides from monkfish swim bladders, and the prepared ACEi peptides, including SEGPK, FDGPY and SPGPW, could serve as natural functional components in the development of health care products to control hypertension.

Antioxidant Peptides from Monkfish Swim Bladders: Ameliorating NAFLD In Vitro by Suppressing Lipid Accumulation and Oxidative Stress via Regulating AMPK/Nrf2 Pathway.

In this study, we investigate the ameliorating functions of QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) from monkfish swim bladders on an FFA-induced NAFLD model of HepG2 cells. The lipid-lowering mechanisms revealed that these five oligopeptides can up-regulate the expression of phospho-AMP-activated protein kinase (p-AMPK) proteins to inhibit the expression of the sterol regulatory element binding protein-1c (SREBP-1c) proteins on increasing lipid synthesis and up-regulating the expression of the PPAP-α and CPT-1 proteins on promoting the ß-oxidation of fatty acids. Moreover, QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) can significantly inhibit reactive oxygen species' (ROS) production, promote the activities of intracellular antioxidases (superoxide dismutase, SOD; glutathione peroxidase, GSH-PX; and catalase, CAT) and bring down the content of malondialdehyde (MDA) derived from lipid peroxidation. Further investigations revealed that the regulation of these five oligopeptides on oxidative stress was achieved through activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to raise the expression levels of the heme oxygenase 1 (HO-1) protein and downstream antioxidant proteases. Therefore, QDYD (MSP2), ARW (MSP8), DDGGK (MSP10), YPAGP (MSP13) and DPAGP (MSP18) could serve as candidate ingredients to develop functional products for treating NAFLD.

Monkfish Peptides Mitigate High Fat Diet-Induced Hepatic Steatosis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic syndrome usually accompanied by fatty degeneration and functional impairment. The aim of the study was to determine whether monkfish peptides (LPs) could ameliorate high-fat diet (HFD)-induced NAFLD and its underlying mechanisms. NAFLD was induced in mice by giving them an HFD for eight weeks, after which LPs were administered in various dosages. In comparison to the HFD control group: body weight in the LP-treated groups decreased by 23-28%; triacylglycerol levels in the blood decreased by 16-35%; and low-density lipoproteins levels in the blood decreased by 23-51%. Additionally, we found that LPs elevated the activity of hepatic antioxidant enzymes and reduced the inflammatory reactions within fatty liver tissue. Investigating the effect on metabolic pathways, we found that in LP-treated mice: the levels of phospho-AMP-activated protein kinase (p-AMPK), and phospho-acetyl CoA carboxylase (p-ACC) in the AMP-activated protein kinase (AMPK) pathway were up-regulated and the levels of downstream sterol regulatory element-binding transcription factor 1 (SREBP-1) were down-regulated; lipid oxidation increased and free fatty acid (FFA) accumulation decreased (revealed by the increased carnitine palmitoyltransferase-1 (CPT-1) and the decreased fatty acid synthase (FASN) expression, respectively); the nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant pathway was activated; and the levels of heme oxygenase-1 (HO-1) and nicotinamide quinone oxidoreductase 1 (NQO1) were increased. Overall, all these findings demonstrated that LPs can improve the antioxidant capacity of liver to alleviate NAFLD progression mainly through modulating the AMPK and Nrf2 pathways, and thus it could be considered as an effective candidate in the treatment of human NAFLD.

Monkfish (Lophius litulon) Peptides Ameliorate High-Fat-Diet-Induced Nephrotoxicity by Reducing Oxidative Stress and Inflammation via Regulation of Intestinal Flora.

BACKGROUND: Renal damage and intestinal flora imbalance due to lipotoxicity are particularly significant in terms of oxidative stress and inflammation, which can be alleviated with bioactive peptides. The monkfish (Lophius litulon) is rich in proteins, which can be used as a source of quality bioactive peptides. This study aimed to examine the protective effect of monkfish peptides on renal injury and their potential role in regulating gut microbiota. METHODS: Monkfish meat was hydrolyzed using neutral protease and filtered, and the component with the highest elimination rate of 2,2-diphenyl-1-picrylhydrazyl was named lophius litulon peptides (LPs). Lipid nephrotoxicity was induced via high-fat diet (HFD) feeding for 8 weeks and then treated with LPs. Oxidative stress, inflammatory factors, and intestinal flora were evaluated. RESULTS: LP (200 mg/kg) therapy reduced serum creatinine, uric acid, and blood urea nitrogen levels by 49.5%, 31.6%, and 31.6%, respectively. Renal vesicles and tubules were considerably improved with this treatment. Moreover, the activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity increased significantly by 198.7%, 167.9%, 61.5%, and 89.4%, respectively. LPs attenuated the upregulation of HFD-induced Toll-like receptor 4 and phospho-nuclear factor-kappa B and increased the protein levels of heme oxygenase 1, nicotinamide quinone oxidoreductase 1, and nuclear factor erythroid 2-related factor 2. The dysbiosis of intestinal microbiota improved after LP treatment. CONCLUSIONS: LPs significantly improve antioxidant activity, reduce inflammatory cytokine levels, and regulate intestinal dysbiosis. Thus, LPs are potential compounds that can alleviate HFD-induced renal lipotoxicity.

Optimization of Extraction of Bioactive Peptides from Monkfish (Lophius litulon) and Characterization of Their Role in H2O2-Induced Lesion.

BACKGROUND: Marine fish meat has been widely used for the extraction of bioactive peptides. This study was aimed to optimize the preparation of monkfish muscle peptides (LPs) using response surface methodology (RSM) and explore the antioxidant activities of <1 kDa LPs. METHODS: Peptides were prepared from the muscles of monkfish (Lophius litulon), and five proteases were tested to hydrolyze muscle proteins. The hydrolysate that was treated using neutrase showed the highest degree of hydrolysis (DH) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities. RESULTS: The optimized conditions were as follows: water/material ratio of 5.4:1, a time span of 5 h, pH of 7.0, enzyme concentration of 2000 U/g, and temperature of 45 °C; the maximum DPPH scavenging activity and DH were 92.861% and 19.302%, respectively. LPs exhibited appreciable antioxidant activities, including DPPH radical, hydroxyl radical, 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) radical, and superoxide anion scavenging activities. LPs attenuated H2O2-related oxidative injury in RAW264.7 cells, reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels. CONCLUSION: We concluded that LPs could be an ideal source of bioactive peptides from monkfish and also have pharmaceutical potential.

Antioxidant Peptides from the Protein Hydrolysate of Monkfish (Lophius litulon) Muscle: Purification, Identification, and Cytoprotective Function on HepG2 Cells Damage by H2O2.

In the work, defatted muscle proteins of monkfish (Lophius litulon) were separately hydrolyzed by pepsin, trypsin, and in vitro gastrointestinal (GI) digestion methods, and antioxidant peptides were isolated from proteins hydrolysate of monkfish muscle using ultrafiltration and chromatography processes. The antioxidant activities of isolated peptides were evaluated using radical scavenging and lipid peroxidation assays and H2O2-induced model of HepG2 cells. In which, the cell viability, reactive oxygen species (ROS) content, and antioxidant enzymes and malondialdehyde (MDA) levels were measured for evaluating the protective extent on HepG2 cells damaged by H2O2. The results indicated that the hydrolysate (MPTH) prepared using in vitro GI digestion method showed the highest degree of hydrolysis (27.24 ± 1.57%) and scavenging activity on a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (44.54 ± 3.12%) and hydroxyl radical (41.32 ± 2.73%) at the concentration of 5 mg protein/mL among the three hydrolysates. Subsequently, thirteen antioxidant peptides (MMP-1 to MMP-13) were isolated from MPTH. According to their DPPH radical and hydroxyl radical scavenging activity, three peptides with the highest antioxidant activity were selected and identified as EDIVCW (MMP-4), MEPVW (MMP-7), and YWDAW (MMP-12) with molecular weights of 763.82, 660.75, and 739.75 Da, respectively. EDIVCW, MEPVW, and YWDAW showed high scavenging activities on DPPH radical (EC50 0.39, 0.62, and 0.51 mg/mL, respectively), hydroxyl radical (EC50 0.61, 0.38, and 0.32 mg/mL, respectively), and superoxide anion radical (EC50 0.76, 0.94, 0.48 mg/mL, respectively). EDIVCW and YWDAW showed equivalent inhibiting ability on lipid peroxidation with glutathione in the linoleic acid model system. Moreover, EDIVCW, MEPVW, and YWDAW had no cytotoxicity to HepG2 cells at the concentration of 100.0 µM and could concentration-dependently protect HepG2 cells from H2O2-induced oxidative damage through decreasing the levels of reactive oxygen species (ROS) and MDA and activating intracellular antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). These present results indicated that the protein hydrolysate and isolated antioxidant peptides from monkfish muscle, especially YWDAW could serve as powerful antioxidants applied in the treatment of some liver diseases and healthcare products associated with oxidative stress.

Optimal Production of Protein Hydrolysates from Monkfish By-Products: Chemical Features and Associated Biological Activities.

The aim of this work was the recovery of protein substrates from monkfish waste (heads and viscera) generated in the on-board processing of this species. Initially, the effect of pH, temperature, and protease concentration was studied on mixtures of a 1:1 ratio (w/v) of monkfish heads/water. The optimal conditions of proteolytic digestion were established at 57.4 °C, pH 8.31, [Alcalase] = 0.05% (v/w) for 3 h of hydrolysis. Later on, a set of hydrolysis at 5L-pH-stat reactor were run under the aforementioned conditions, confirming the validity of the optimization studies for the head and viscera of monkfish. Regarding the chemical properties of the fish protein hydrolysates (FPH), the yield of digestion was higher than 90% in both cases and the degrees of hydrolysis and the soluble protein content were not especially large (<20% and <45 g/L, respectively). In vitro digestibility was higher than 90% and the percentage of essential amino acids ranged from 40 to 42%. Antioxidant activities were higher in viscera FPH, and antihypertensive ability was superior in head FPH. The values of number average molecular weights (Mn) of monkfish hydrolysates were 600 Da in the viscera and 947 Da in the head. The peptide size distribution, obtained by size-exclusion chromatography, indicated that the largest presence of peptides below 1000 Da and 200 Da was observed in the viscera FPH.

Optimization of phosphate recovery from monkfish, Lophius vomerinus, processing by-products and characterization of the phosphate phases.

BACKGROUND: Fish-processing by-products represent an increasing proportion of wastes globally. Valorizing offers a sustainable alternative by harnessing high-value products through process development. This study aimed to develop and optimize a demineralization process to recover minerals from fish bones with subsequent recovery of phosphates from the resulting solution. RESULTS: The demineralization process was optimized under the following conditions: 5% H3 PO4 concentration (v/v), four extractions and solvent to feed ratio (v/w) of 5:1 at ambient temperature of 17 °C. This resulted in an ossein containing 2.0 ± 1.2 g kg-1 DM ash and 71.5 ± 3 g kg-1 DM hydroxyproline and mineral liquor. The phosphate precipitation from the mineral liquor was further optimized resulting in > 99% total P recovery at 75 °C reaction temperature and 1 mol L-1 Ca(OH)2 to mineral liquor ratio(v/v) of 0.95:1 for a reaction time of 17 min, per 150 mL starting mineral liquor. The precipitate contained 215.2 ± 3.0 g kg-1 DM dicalcium phosphate dihydrate (DCPD) with a net contribution of 17.2% P from the fishbones, and 25 ± 0.2 g DM of octacalcium phosphate (OCP) was precipitated from 150 mL starting mineral liquor at 25 °C reaction temperature, (1.2:1) 1 mol L-1 Ca(OH)2 to mineral liquor ratio (v/v) and reaction time of 17 min. The X-ray spectra confirmed the DCPD structure and Fourier transform infrared spectroscopy (FTIR) spectra indicated OCP precipitation. CONCLUSION: This work successfully illustrated the recovery of minerals from fish bones and the subsequent production of different high-quality phosphates from fish-processing by-products, thus indicating a potential source for high-value products. © 2018 Society of Chemical Industry.

Goosefish Lophius americanus fecundity and spawning frequency, with implications for population reproductive potential.

To improve knowledge of goosefish Lophius americanus' reproductive biology, females were collected during 2009-2012 from the Mid-Atlantic Bight shelf region of the U.S. east coast. Batch fecundity increased with total length (LT ), from 229 100 to 2 243 300 mature oocytes per female (LT range: 55·5-112 cm; n = 54). This estimate of fecundity at LT is lower than one derived from a sample collected during 1982-1985. Examination of whole oocyte diameters in different months indicated that L. americanus is a serial spawner, releasing more than one egg veil per spawning season, as suspected or observed for other Lophius species. Seasonality of spawning was evident from whole oocytes and gonad histology, and from larval fish surveys spanning the U.S. north-east shelf, and confirmed a protracted (c. 6 months) spawning period. Peak spawning activity progressed northward from spring to autumn. The population-level implications of these results were explored by estimating population reproductive potential (PRP ), which considered the value of both current and future per capita reproduction using decade-specific age structure and fecundity at length. PRP is now more than 50% lower compared with the historical period (1982-1985), a result of the lower proportions of large females and reduced fecundity across all sizes. Mechanisms that could explain this loss of stock productivity are fishing-induced size-age truncation or regime shifts in egg production caused by changes in energy density of common forage species.

The chorion ultrastructure of ova of Lophius spp.

The chorion surface ultrastructure of unfertilized eggs of black anglerfish Lophius budegassa and white anglerfish Lophius piscatorius was examined by scanning electron microscopy. Species-specific differences were observed.

Inferring fish behaviour at the trawl mouth from escape location.

In this study, we used escape location underneath the trawl to understand groundfish herding behaviour at the trawl mouth. Three collecting bags (port, center, starboard) were mounted under the trawl and behind the footgear to collect escapees. The escape-at-length of species that escaped into the center bag were compared to the two wing bags to infer fish response behaviour, herding behaviour, and swimming capacity at the trawl mouth. For roundfish, smaller-sized individuals escaped more in the center for both Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus), <20 and 11 cm, respectively, indicating that larger-sized fish were to a greater extent seeking to escape under the trawl at the wings, vs small fish being herded to the center and likely overrun due to reduced swimming capacity. For flatfish and monkfish (Lophius piscatorius), results varied. European plaice (Pleuronectes platessa), American plaice (Hippoglossoides platessoides), and monkfish were caught most often in the wings, though not significantly for American plaice. Catches of dab (Limanda limanda) between 18 and 27 cm were significantly higher in the center, with no difference for smaller and larger individuals. The differences between fish escape location likely result from a combination of varying herding behaviour, size, and swimming capacity. Here, we were able to show how these size-dependent behaviours relate to fish response behaviour, escape behaviour, size, and likely swimming capacity.

Fish Liver Discards as a Source of Long-Chain Omega-3 Polyunsaturated Fatty Acids.

The intake of omega-3 polyunsaturated fatty acids (PUFA) of the average consumer is generally low, and products such as fish oils high in omega-3 PUFA have become popular dietary supplements. There is a need for more sources of omega-3 PUFA to cover the increasing demand. This study investigated whether livers from different lean fish species could be a potential new source of oils rich in omega-3 PUFA. The seasonal variation in lipid content, fatty acid composition, peroxide value and free fatty acid content (FFA) of livers from cod, hake, ling, coalfish and monkfish was determined, and the effect of storage conditions on the fishing vessel (ice vs frozen) was studied. Generally, the lipid content and composition of the livers from the five fish species varied similarly during the two years of the sampling period, with significantly lower values in spring (March, April) and higher values in fall (October, November). Storage conditions were found to have no significant effect on the quality and oil composition. Monkfish livers were less suitable for production of omega-3 oil due to their lower lipid and EPA content as well as higher FFA levels. Coalfish had higher fluctuations in oil composition during the sampling period, which potentially makes a standardised quality difficult to obtain. Cod, hake and ling were the most suitable species for fish liver oil production.

The effect of capture and handling stress in Lophius americanus in the scallop dredge fishery.

Capture and handling stress studies are considered a primary research priority, particularly for species and fisheries where discard rates are high, and/or for overfished stocks and species of concern. Lophius americanus, a commercially valuable finfish in New England, constitutes the second highest bycatch species within the sea scallop dredge fishery. Despite its commercial importance, no data exists on the capture and handling stress of monkfish for any gear type. Given these shortcomings, our goals were to evaluate the stress response of monkfish captured in scallop dredge gear by evaluating physical, behavioural and physiological responses to scallop fishing practices. While 80% of monkfish displayed little to no physical trauma, behavioural and physiological assessment indicated high levels of stress, especially as air exposure and tow duration increased. This finding suggests that the manifestation of stress in monkfish may be a cryptic response necessitating further research in addition to estimates of post-release mortality rates to appropriately advise fisheries management regarding the mortality of monkfish bycatch in the sea scallop fishery.

Mercury concentrations in muscles and liver tissues of Cape monkfish (Lophius vomerinus) from the Northern Benguela, Namibia.

Cape monkfish (Lophius vomerinus) is one of the long-lived species and top predators in the northern Benguela region. Studies on bioaccumulation of mercury (Hg) in cape monkfish are limited. This study compared the total Hg concentration between monkfish muscle and liver tissue; and related the monkfish total Hg concentrations to fish body size and capture locations (depth and latitude). Monkfish specimens (n = 529) were collected from 2016 to 2018. The mean total Hg concentration was 0.126 ±â€¯0.005 mg/kg in muscle tissues and 0.106 ±â€¯0.005 mg/kg in liver tissues. No significant differences were observed between total Hg concentrations of muscles and liver tissues. Differences in Hg concentrations between monkfish length classes were observed. No significant correlation was found between total Hg concentrations and latitude. A significant increase of total Hg concentration with the depth was observed. The concentrations of Hg were below the World Health Organization (WHO limits for fish (0.5 mg/kg).