Cod Bones Research Articles

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1–2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated “GE” motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

Lactic Acid-Based Natural Deep Eutectic Solvents to Extract Bioactives from Marine By-Products.

Natural deep eutectic solvents (NaDES) were used to extract bioactive compounds from marine by-products: codfish bones, mussel meat, and tuna vitreous humor. NaDES were prepared using natural compounds, including lactic acid (Lac), fructose (Fru), and urea (Ur), and were characterized to define their physicochemical properties, including the viscosity, density, surface tension, and refractive index. FTIR and NMR analysis confirmed the presence of intermolecular hydrogen bonding in NaDES. The extracts obtained using these NaDES were characterized to define their composition. Results demonstrated that the extract’s composition differed highly, depending not only on the DES used, but also on the structure and composition of the raw material. Proteins and lipids were mainly present in extracts obtained from mussels, while ash content was highest in the extracts obtained from codfish bones. The biocompatibility of NaDES and the soluble fractions (SF) of the raw materials in NaDES was evaluated, and it was possible to conclude that the soluble ingredients obtained from the raw materials improved the biocompatibility of NaDES.

Study on Optimization of Enzymatic Hydrolysis of Fish Bone Paste and Flavor Characteristics of Hydrolysates

Taking the ultra-fine fish bone paste prepared from cod bones as the object, the conditions of enzymatic hydrolysis of fish bone paste were optimized by using the single factor experiment and response surface design based on the degree of hydrolysis as an indicator. Meanwhile, the changes of flavor characteristics of fish bone paste and its hydrolysates were analyzed by employing electronic nose, electronic tongue, gas-phase ion migration chromatography(GC-IMS), and free amino acid determination. The results showed that the optimal enzymatic hydrolysis conditions were obtained at hydrolysis time of 5 hours, enzyme addition of 1.0%, material-liquid ratio of 1:1 by using flavor protease, yield 43.8% of hydrolysis degree. The electronic nose analysis results showed that the aromatic substances in the hydrolysate increased, while the content of undesirable compounds such as alkanes and ammonia decreased. The results of electronic tongue analysis showed that the acidity of the hydrolysate was increased, while the bitterness was reduced, and the overall taste was good and rich. There were 29 volatile compounds of hydrolysate and fish bone paste were detected by GC-IMS. In fish bone paste, the main volatile flavor substances were aldehydes and ketones, and the main flavor substances in the hydrolysate were ketones and esters. These types and contents of volatile flavor substances were significantly different. The hydrolysate increased the content of flavored amino acids and the hydrolysate had a good flavor.

Preparation and Characteristics of Low-Bitter Peptides from the Enzymatic Hydrolysate of Cod Bone

In this study, cod bones were used as raw material. The bitterness of cod bone hydrolysate was reduced by controlling the degree of hydrolysis, and the mechanism of peptide and free amino acids in the hydrolysate on bitterness was analyzed. The results showed that the degree of hydrolysis reached 7.48% and the bitterness value was 5.80 through mild hydrolysis for 2 h. The peptides were mainly distributed between 1500 and 2000 Da. In addition, the precipitate was subjected to high-pressure cooking and heat treatment (121 °C, 30 min) to make the surface of the fish bone loose after the first enzymolysis. The degree of hydrolysis reached 49.24% and the bitterness value was 6.03 through extensive hydrolysis for 2 h. The peptides were mainly distributed between 500 and 1000 Da. Free amino acids were significantly higher than the content after only the first hydrolysis. By controlling the degree of hydrolysis to limit the production of bitterness, the preparation of this low-bitterness enzymatic peptide laid the foundation for its application in food.

High stability of bilayer nano-emulsions fabricated by Tween 20 and specific interfacial peptides

To ensure emulsions to be continuously stable, the hydrolysates recovered from cod bones by papain acted as a natural surfactant to synthesize high-stability bilayer nano-emulsions. As assisted by Tween 20, the average diameter of synthesized nano-emulsion with enzymatic hydrolysate could exhibit stability between 300–400 nm under a broad range of pH (4–8), temperatures (30–90 °C) and salt concentration (25–250 mM). With the addition of the hydrolysates, the rate of increase of the TBARS value in the emulsion decreased. Moreover, the bilayer structure of the nano-emulsion was characterized under an atomic force microscopy and a cryo-scanning electron microscopy. Nano-LC-Q-TOF-MS was adopted to primarily identify peptides that contained hydrophobic and hydrophilic amino acids at the emulsion interface. Besides, the absorbed peptides on the interface of emulsion enhanced the stability of emulsion lipid oxidation.

Films of chitosan and natural modified hydroxyapatite as effective UV-protecting, biocompatible and antibacterial wound dressings

Chitosan is a natural polysaccharide widely used in biomedicine, for instance for wound dressing. Hydroxyapatite is a very bioactive calcium phosphate which, if modified with an appropriate element (iron Fe), can also have UV-absorbing properties. In this work, we report the study of films of chitosan incorporated with iron-modified hydroxyapatite of natural origin (from cod fish bones); this combination led to an innovative chitosan-based material with excellent and advanced functional properties. The films showed very high UV absorption (Ultraviolet Protection Factor (UPF) value higher than 50). This is the first time that a chitosan-based material has shown such high UV protection properties. The films also showed to be non-cytotoxic, and possessed antimicrobial activity towards both Gram-positive and negative strains. Their mechanical properties, optimised with an experimental design approach, confirmed their potential use as multifunctional wound dressing, capable of reducing bacterial infections and, at the same time, protecting from UV light.

Extraction of hyaluronic acid and chondroitin sulfate from marine biomass for their application in the treatment of the dry eye disease

AbstractPurposeTear film instability, hyperosmolarity and neurosensory abnormality are the key etiological contributors to the pathophysiological process of the Dry Eye Disease (DED).1 Hyaluronic acid (HA) and Chondroitin sulfate (CS) have shown improvements in the corneal epithelial barrier in patients with DED.2 Moreover they are used in pharmaceutical applications due to their biocompatibility, viscoelasticity, lubricity and immunostimulatory effects. In this study, they are extracted from marine wastes and will be evaluated as possible DED treatment using natural, green and novel Deep Eutectic Solvents (DES) to replace time‐consuming and expensive conventional methods and maintaining the quality and purity of the isolated HA and CS. Natural compounds such as menthol and trehalose, are used in DES preparation and have shown to be effective in DED treatment.3,4MethodsHA and CS are isolated from marine wastes using conventional methods and DES as low‐cost alternative technique. DES are prepared by heating and mixing the components at certain ratios. They are mixed with the marine sources at specific extraction conditions. The extracts are then enzymatically hydrolyzed and analyzed by HPLC and capillary electrophoresis to quantify HA and CS.ResultsHA and CS are extracted from mussels, codfish bones and furtherly tuna eyeballs and shark fins using DES. Several DES were tested such as menthol:borneol, menthol:camphor, trehalose:glycerol, and significant amounts of HA and CS were extracted with high specificity. For example, 37.92 µg/gdry codfish bones CS disaccharide (ΔHexAβ1‐3GalNAc4S) were extracted using menthol:borneol.ConclusionDES are effective compounds for the extraction of valuable biopolymers HA and CS. The isolated HA and CS, as well as their therapeutic mixture with DES will be studied in vitro on conjunctival and corneal epithelial human cell lines to assess their toxicity, biocompatibility, muco‐adhesion and anti‐oxidant effects for the treatment of the DED.References Craig, Jennifer P., et al. “TFOS DEWS II report executive summary.” The Ocular Surface 15.4 (2017): 802‐812. Limberg, Michael B., et al. “Topical application of hyaluronic acid and chondroitin sulfate in the treatment of dry eyes.” American journal of ophthalmology 103.2 (1987): 194‐197. Arita, Reiko, et al. “Effects of a warm compress containing menthol on the tear film in healthy subjects and dry eye patients.” Scientific reports 7 (2017): 45848. Fondi, Klemens, et al. “Effect of Hyaluronic Acid/Trehalose in Two Different Formulations on Signs and Symptoms in Patients with Moderate to Severe Dry Eye Disease.” Journal of ophthalmology 2018 (2018).

Increased UV absorption properties of natural hydroxyapatite‐based sunscreen through laser ablation modification in liquid

AbstractSunfilters based on hydroxyapatite (HAp) and iron‐containing compounds (Fe2O3 and calcium iron phosphates) are of increasing interest, as they show UV absorption without generating health endanger free radicals (usually observed when other inorganic sunscreens are used). In this paper, laser ablation of solids in liquids has been applied to improve the UV absorption properties of a HAp based Fe‐containing sunscreen powder derived from cod fish bones. Two different laser wavelengths were explored (532 and 1064 nm, green and infrared, respectively); an improved experimental device was used, to allow a fine control of the volume of the irradiated particles. Results show an increased UV absorbance for the laser‐treated powders in comparison with the untreated ones; this can be explained considering the smaller particle size and increased surface area; the higher iron concentration in the powders may also be determinant. Enhanced absorption was also observed in the near‐infrared range, making the powders even more suitable for sunscreen applications. The green laser was more effective than the infrared one. Overall, laser ablation showed to be a powerful technique to control the size of the sunscreen particles and tailor their optical properties.

Effects of lipid extraction and different collagen extraction methods on archaeological fish bones and its implications for fish bone diagenesis

Gelatin extracted from archaeological fish bones typically exhibits relatively high C/N ratios, presumed to be caused by contamination with lipids or humic substances. The effects of lipid extraction and different collagen extraction methods applied has been studied on modern fish bones but has never been studied systematically on archaeological specimens because of taphonomic and experimental reasons. In this study, the effects of lipid extraction and order of NaOH treatment in collagen extraction method on carbon and nitrogen stable isotope analysis of archaeological fish bone (n = 30) excavated from the Hamanaka 2 site, Hokkaido, Japan (approximately 8th BC–10th AD) is investigated. Gelatin extracted from the same fish bone subsamples with or without lipid extraction procedure indicates neither significant nor systematic differences in carbon and nitrogen stable isotope ratios, elemental concentrations, C/N ratios, and yield. However, reproducibility of stable isotope ratios and elemental concentrations decrease in gelatin extracted from poorly-preserved cod bones (<3.5% yield). Gelatin extracted from archaeological fish bones may contain humic contaminants, and its effect becomes greater in gelatin with lower extraction yield. Although there is no significant change in the stable isotope and C/N ratios, change in atomic concentration of carbon and nitrogen suggests that the purity of extracted gelatin increases when NaOH treatment is applied after decalcification. Because this is only a study from one archaeological site, further case studies that evaluate lipids and diagenesis in fish bones are required.

A millennium of north-east Atlantic cod juvenile growth trajectories inferred from archaeological otoliths.

Archaeological excavations of historical fishing sites across the North Atlantic have recovered high quantities of Atlantic cod (Gadus morhua) bones. In the current study we use Atlantic cod otoliths from archaeological excavations of a historical fishing sites in north-west Iceland, dated to AD 970 –AD 1910 to examine historical growth trajectories of cod. No large scale growth variations or shifts in growth patterns were observed in the current chronologies, supporting the stability of historical Atlantic cod growth trajectories. The most significant variation in growth patterns was consistent with those that have been observed in recent times, for example, reduced early juvenile growth during periods of colder ocean temperature. The current results represent a high resolution chronological record of north-east Atlantic cod growth, greatly increasing the prior temporal range of such data, thereby providing a valuable baseline for a broad range of studies on Atlantic cod growth.

Biphasic apatite-carbon materials derived from pyrolysed fish bones for effective adsorption of persistent pollutants and heavy metals

Biphasic apatite-carbon biochar-type materials were prepared from pyrolysed cod fish bones and were assessed for the adsorption of persistent organic pollutants (pharmaceuticals diclofenac and fluoxetine), and heavy metals (Pb(II)).The materials, prepared with a simple pyrolysis process at different temperatures (200–1000°C), were characterised with XRD, FTIR, Raman and SEM. Results showed that the pyrolysis temperature had a significant effect on the features/composition of the materials: up to 800 °C, carbonate apatite Ca10(PO4)6(CO3) was the main component, while for higher temperatures oxyapatite Ca10(PO4)6O was the dominant phase. Graphitic carbon was also detected.The mixed apatite-carbon products (bone char) exhibited high adsorption efficiency.Graphite carbon was the main adsorber for the pharmaceuticals, the best performing material being that pyrolysed at 1000°C. Xm values of 43.29 and 55.87mg/g were observed (Langmuir fitting), while KF values of 5.40 and 12.53 (mg/g)(L/mg)nF were obtained with the Freundhlich model (diclofenac and fluoxetine respectively). This is the first time that a biochar-like material has been used for fluoxetine adsorption. For Pb (II), the powder pyrolysed at 600°C was the most effective, with the apatite playing a key role (Xm=714.24mg/g).This work shows that a by-product of the fish industry could be converted into efficient materials for environmental remediation; according to the pyrolysis conditions, powders effective in the removal of either organics or heavy metals can be obtained. Moreover, with pyrolysis at intermediate temperatures, materials capable of adsorbing both kinds of pollutants can be produced, even if less efficient.

Luminescent calcium phosphate bioceramics doped with europium derived from fish industry byproducts

AbstractThe recovery of valuable compounds from byproducts is at present a priority topic for social, environmental, and economic reasons. In this work we report for the first time the preparation of luminescent calcium phosphate (CaP) bioceramics from waste codfish bones. Codfish bones were treated in aqueous Eu(NO3)3 solutions of different concentrations, followed by thermal treatment at either 700°C or 1100°C. The resulting materials consisted of hydroxyapatite (HAp), β‐tricalcium phosphate (β‐TCP) and a small amount of Eu2O3. Chemical and structural characterization showed that the Eu ions were successfully introduced into the bones and, hence, in the thermally treated CaP phases obtained from them, although mainly in the β‐TCP lattice. Photoluminescence investigation revealed that all the materials are luminescent, with samples treated at 1100°C having more intense luminescence than those prepared at 700°C. In vitro evaluation of cell‐materials interaction indicated that all the samples displayed good cytocompatibility toward osteoblast cells. This work demonstrates that a simple and effective process, employing Eu as a dopant, can convert fish industry byproducts into highly valuable luminescent CaP bioceramics, having potential applications in biology and medicine for bio‐imaging.

Effect of preparation and processing conditions on UV absorbing properties of hydroxyapatite-Fe2O3 sunscreen

The development of innovative, safe and non-photocatalytic sunscreens is urgently needed, as it is essential to have sunscreen filters offering appropriate UV protection without damaging the environment and/or generating free radicals when in contact with the skin.Hydroxyapatite (Ca10(PO4)6(OH)2, HAp) when substituted with iron has UV protection properties and is not photocatalytic; HAp was used to make a sunscreen filter by treating cod fish bones in an iron-containing solution, and then calcining them at 700°C. Here we present a systematic and advanced study on this material, to obtain a sunscreen with improved UV absorbing properties. Bones were treated with three different iron salts - Fe(II) chloride, Fe(II) lactate and Fe(III) nitrate - under various pH conditions.Results showed that Fe(II) chloride in basic pH led to the most effective iron inclusion. High energy ball milling or ultrasound were investigated to increase surface area and corresponding UV absorption; high energy ball milling treatment led to the best optical properties.The optimum powders were used to formulate UV protection creams, which showed Sun Protection Factor (SPF) values significantly superior to the control cream (up to 4.1). Moreover the critical wavelength (λcrit) was >370nm (388–389nm) and UVA/UVB ratios were very close to 1. With these properties these sunscreens can be classified as broad UV protectors.Results also showed that combining these powders with other sunscreens (i.e. titanium dioxide), a synergic effect between the different components was also observed. This investigation showed that HAp-based sunscreens of marine origin are a valid alternative to commercial products, safe for the health of the customers and, being non-photocatalytic, do not pose a threat to the environment.

Photodegradation of pharmaceutical persistent pollutants using hydroxyapatite-based materials

Pharmaceutical persistent pollutants pose a serious threat to the environment. The aim of this study was to use, for the first time, hydroxyapatite-based biomaterials as photocatalysts to degrade micropollutants. Diclofenac and fluoxetine were selected for these initial tests. Hydroxyapatite (Ca10(PO4)(OH)2, HAp) is one of the most commonly used biomaterials/bioceramics, being a major constituent of bone. In this work sustainable HAp-based materials of marine origin, obtained from cod fish bones, were used; these photocatalysts were previously fully studied and characterised. Both single-phase HAp and HAp–titania multicomponent materials (1 wt% TiO2) were employed as UV light photocatalysts, the latter showing better performance, indicated by higher degradation rates of both compounds. The HAp-titania photocatalyst showed excellent degradation of both persistent pollutants, the maximum degradation performance being 100% for fluoxetine and 92% for diclofenac, with pollutant and photocatalyst concentrations of 2 ppm and 4 g/L, respectively. Variations in features such as pollutant and photocatalyst concentrations were investigated, and results showed that generally fluoxetine was degraded more easily than diclofenac. The photocatalyst's crystallinity was not affected by the photodegradation reaction; indeed the material exhibited good photostability, as the degradation rate did not decrease when the material was reused. Tests were also performed using actual treated wastewater; the photocatalyst was still effective, even if with lower efficiency (−20% and −4% for diclofenac and fluoxetine, respectively). TOC analysis showed high but incomplete mineralisation of the pollutants (maximum 60% and 80% for DCF and FXT, respectively).

Reuse and recycling in construction: novel photocatalyticnano-mortars from valorised industrial wastes

Production of novel eco-composite nano-mortars for architectural finishing, designed to combat environmental pollution affecting building shells, is reported. Specimens consist of a natural hydraulic lime mortar matrix doped with various kinds of nanophotocatalysts, produced from valorized industrial wastes: TiO2-hexaferrite from Fe3O2, and Ti(SO4)2-hydroxyapatite from Atlantic codfish bones. Samples were characterized and their photocatalytic activity was evaluated. Analysis showed that industrial wastes may be usefully reused to produce a new generation of sustainable, cheap and multifunctional novel building materials.

Evaluation of CT capability for the detection of thin bone structures

Abstract Evaluation of CT capability for the detection of thin bone structures by means of modeling small bone structures and comparing with quantitative measurements. Knowledge of how thin bone structures can be or of what low density to be detectable in clinical CT images can increase the diagnostic competence to diagnose various diseases. Correlation between size and density of detectable bone structures is the key for many diagnosis purposes. This paper describes the use of clinical 64 detector scanner to image the skeletons of codfish and salmon species. Fish skeletons, such as codfish and salmon, have petite structures and lower bone density than humans. Bone structures were segmented out of the image data and 3D models of their skeletons developed. Evaluation was done by means of comparing quantitative measurements of selected bones to parameters observed from the model. Results show the limits where thin bones and of low density disappear from the clinical CT. It shows ability to reconstruct closely the diameter of the codfish bones but to a less extent the small bones of the salmon.

The globalization of naval provisioning: ancient DNA and stable isotope analyses of stored cod from the wreck of the Mary Rose, AD 1545.

A comparison of ancient DNA (single-nucleotide polymorphisms) and carbon and nitrogen stable isotope evidence suggests that stored cod provisions recovered from the wreck of the Tudor warship Mary Rose, which sank in the Solent, southern England, in 1545, had been caught in northern and transatlantic waters such as the northern North Sea and the fishing grounds of Iceland and Newfoundland. This discovery, underpinned by control data from archaeological samples of cod bones from potential source regions, illuminates the role of naval provisioning in the early development of extensive sea fisheries, with their long-term economic and ecological impacts.

Detection of Fish Bones in Cod Fillets by UV Illumination

The presence of fish bones is now regarded as an important hazard in fishery products, and there is increasing demand for new analytical techniques to control it more effectively. Here, the fluorescent properties of cod bones under UV illumination were investigated, and the maximal wavelengths for excitation and emission were determined to be 320 nm and 515 nm, respectively, demonstrating significantly different fluorescence characteristics and much higher fluorescence intensity compared to those of fillet muscles. Based on the results, UV fluorescence-assisted candling for the detection of bones in fishery products was developed for the first time. Using cod fillets as samples, the detection ratio of this technique was calculated as 90.86%, significantly higher than that of traditional candling under daylight (76.78%). Moreover, the working efficiency of the new technique was about 26% higher than that of the traditional method. A UV fluorescence imaging framework was also developed, and a method for automatic identification of the fish bones in the cod fillets based on the linear discriminant analysis proposed by Fisher was preliminarily realized, but the detection ratio was demonstrated to be relatively poor compared to those of candling techniques. These results allow us to suggest UV-based methods as new and promising approaches for routine monitoring of bones in fishery products.

Hydroxyapatite-based materials of marine origin: a bioactivity and sintering study.

Single phase hydroxyapatite (HAp) and biphasic material hydroxyapatite/β-tricalcium phosphate (HAp/β-TCP) were obtained from a marine source (Atlantic cod fish bones). Here we report a study on the biological properties of these materials, including cytotoxicity, bioactivity and haemocompatibility. Results showed that the materials are not cytotoxic, neither in their powder nor in pellet form; indeed growth of Saos-2 cells was comparable to that of commercial. The haemolysis rate was lower than 2%; hence the materials can be classified as non-haemolytic. Moreover, when immersed in Simulated Body Fluid (SBF), crystal formation was observed on the surface of both materials. The sintering behaviour of the samples was also studied; both powders showed very high sinterability (density higher than 95% of the theoretical value). Overall, these results confirm the suitability of these materials for biomedical applications.

Testing the long-term stability of marine isoscapes in shelf seas using jellyfish tissues

Maps of the spatial distribution of stable isotope ratios across wide geographic areas (isoscapes) are increasingly used to study mechanisms of nutrient flux, movements of animals, and to improve trophic information derived from stable isotope analyses. Isoscapes are usually constructed from reference samples collected from known geographic positions, a time consuming and costly process. In this study, we test the temporal stability of isoscapes of carbon and nitrogen isotopes across the North Sea over a ten-year period. Using jellyfish tissues as reference organisms, we show that hydrodynamic and biogeochemical processes controlling the distribution of carbon and nitrogen isotope values, and thus that the underlying isoscapes, are temporally stable. Remarkably, broad geographic variations in stable carbon and nitrogen isotope distributions across the North Sea are consistent with previously published variations seen in archaeological cod bones from the ninth to seventeenth centuries, despite dramatic changes in land use in the past 1,000 years. Stable isotope-based studies of trophic interactions or movements in animals with lifespans of a year or more in shelf ecosystems can consequently be referenced to previously published isoscape models, and do not require construction of temporally explicit isotope baseline corrections. Scyphomedusan jellyfish are excellent reference organisms for ecological stable isotope analyses in pelagic ecosystems, due to their widespread distributions, well-defined life histories, and fast tissue growth.