Potential Biomedical Applications of Collagen Filaments derived from the Marine Demosponges Ircinia oros (Schmidt, 1864) and Sarcotragus foetidus (Schmidt, 1862)

Collagen filaments derived from the two marine demosponges Ircinia oros and Sarcotragus foetidus were for the first time isolated, biochemically characterised and tested for their potential use in regenerative medicine. SDS-PAGE of isolated filaments revealed a main collagen subunit band of 130 kDa in both of the samples under study. DSC analysis on 2D membranes produced with collagenous sponge filaments showed higher thermal stability than commercial mammalian-derived collagen membranes. Dynamic mechanical and thermal analysis attested that the membranes obtained from filaments of S. foetidus were more resistant and stable at the rising temperature, compared to the ones derived from filaments of I. oros. Moreover, the former has higher stability in saline and in collagenase solutions and evident antioxidant activity. Conversely, their water binding capacity results were lower than that of membranes obtained from I. oros. Adhesion and proliferation tests using L929 fibroblasts and HaCaT keratinocytes resulted in a remarkable biocompatibility of both developed membrane models, and gene expression analysis showed an evident up-regulation of ECM-related genes. Finally, membranes from I. oros significantly increased type I collagen gene expression and its release in the culture medium. The findings here reported strongly suggest the biotechnological potential of these collagenous structures of poriferan origin as scaffolds for wound healing.     

Application of the Gadidae Fish Processing Waste for Food Grade Gelatin Production

Waste from fish cutting (heads, swim bladders, fins, skin, and bones) is a high-value technological raw material for obtaining substances and products with a wide range of properties. The possibility of using waste from cutting fish of the Gadidae family: the Alaska pollock (Gadus chalcogrammus) and the Pacific cod (Gadus macrocephalus), processed in the coastal zone, is scientifically substantiated. In this work, a technology has been developed for processing accumulated waste from fish cutting in order to obtain fish gelatin, which is characterized by high protein content (more than 80.0%) and a full set of essential and nonessential amino acids. We studied the quality of fish gelatin obtained from wastes from cutting the fish of the Gadidae family. The possibility of using fish gelatin as a component of fish products is shown; the dose of its introduction into the fish products is substantiated. The data obtained made it possible to recommend the use of fish processing waste products as a gelling component and a source of amino acids in multicomponent food systems.     

Thermal transition properties of hoki (Macruronus novaezelandiae) and ling (Genypterus blacodes) skin collagens: implications for processing

Hoki (Macruronus novaezelandiae) and ling (Genypterus blacodes) are cold-water fish caught in New Zealand waters. Their skins are a major component of the post-processing waste stream. Valuable products could be developed from the skins, as they are primarily composed of collagen, which has many commercial applications. We prepared acid soluble collagens (ASC) from hoki and ling skins, and analyzed their thermal denaturation properties using a Rapid Visco™ Analyzer. At slower heating rates the denaturation temperature (TD) of hoki and ling collagens decreased. This result is consistent with the model of irreversible rate kinetics for the denaturation of collagen. We determined the effects of solvents that disrupt hydrogen bonding on ASC stability. Increasing concentrations of urea from 0.1 M to 1.0 M and acetic acid from 0.1 M to 0.5 M decreased TD. This resulted from the effects of these reagents on the hydrogen bonds that stabilize the collagen triple helix.     

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste, direct quantitative PCR (qPCR) kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study. A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories. Compared with 2 commercial direct qPCR kits, conditions of DNA releasing procedure and PCR amplification were optimized. Element screening was performed at the initial step of genetically modified (GM) ingredient testing procedure via direct qPCR. GM event identification was carried out in positive samples by initial screening. Totally 5 screening elements (P–35S, T-NOS, Cp4-epsps, bar and pat) for soybean materials and 6 screening elements (P–35S, T-NOS, NPTII, Cry1Ac, bar and pat) for cotton samples were detected. In GM event identification, MON531 and MON1445 were found in cotton materials. Results were further confirmed by real-time PCR with DNA extraction and purification. The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.     

Production,Characterization and Biocompatibility of Marine Collagen Matrices from an Alternative and Sustainable Source: The Sea Urchin Paracentrotus lividus

Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30–400 nm and mesh < 2 μm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.     

Affinity Purification of Angiotensin Converting Enzyme Inhibitory Peptides from Wakame (Undaria Pinnatifida) Using Immobilized ACE on Magnetic Metal Organic Frameworks

Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from marine organism have shown a blood pressure lowering effect with no side effects. A new affinity medium of Fe₃O₄@ZIF-90 immobilized ACE (Fe₃O₄@ZIF-90-ACE) was prepared and used in the purification of ACE inhibitory peptides from Wakame (Undaria pinnatifida) protein hydrolysate (<5 kDa). The Fe₃O₄@ZIF-90 nanoparticles were prepared by a one-pot synthesis and crude ACE extract from pig lung was immobilized onto it, which exhibited excellent stability and reusability. A novel ACE inhibitory peptide, KNFL (inhibitory concentration 50, IC₅₀ = 225.87 μM) was identified by affinity purification using Fe₃O₄@ZIF-90-ACE combined with reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry. Lineweaver–Burk analysis confirmed the non-competitive inhibition pattern of KNFL, and molecular docking showed that it bound at a non-active site of ACE via hydrogen bonds. This demonstrates that affinity purification using Fe₃O₄@ZIF-90-ACE is a highly efficient method for separating ACE inhibitory peptides from complex protein mixtures and the purified peptide KNFL could be developed as a functional food ingredients against hypertension.     

Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal

Calli were obtained from leaf, cotyledon and internode explants of in vitro-grown plants of Indian cultivar of Withania somnifera in MS medium supplemented with 2, 4-D (2.0 mg l⁻¹) and Kinetin (0.2 mg l⁻¹). The brown, semi-friable callus (500 mg FW) derived from leaf explants produced higher number of primary adventitious roots (9 roots/callus) in half strength MS medium fortified with IBA (0.5 mg l⁻¹) and NAA (0.1 mg l⁻¹). The primary adventitious roots with an inoculum mass of 15 g FW were cultured for 6 weeks in the same medium for secondary adventitious root proliferation. Elicitation of abiotic elicitor, aluminium chloride at 10 mg l⁻¹ at the end of 4 weeks culture with 4 h exposure time enhanced withanolides productivity. Under similar culture conditions, the biotic elicitor, chitosan at 100 mg l⁻¹ stimulated higher production of all withanolides when compared to aluminium chloride treatment. This is the first report on the use of callus-derived adventitious root culture for the enhanced production of withanolides upon chitosan elicitation.     

Biofunctionality of Enzymatically Derived Peptides from Codfish (Gadus morhua) Frame: Bulk In Vitro Properties,Quantitative Proteomics,and Bioinformatic Prediction

Protein hydrolysates show great promise as bioactive food and feed ingredients and for valorization of side-streams from e.g., the fish processing industry. We present a novel approach for hydrolysate characterization that utilizes proteomics data for calculation of weighted mean peptide properties (length, molecular weight, and charge) and peptide-level abundance estimation. Using a novel bioinformatic approach for subsequent prediction of biofunctional properties of identified peptides, we are able to provide an unprecedented, in-depth characterization. The study further characterizes bulk emulsifying, foaming, and in vitro antioxidative properties of enzymatic hydrolysates derived from cod frame by application of Alcalase and Neutrase, individually and sequentially, as well as the influence of heat pre-treatment. All hydrolysates displayed comparable or higher emulsifying activity and stability than sodium caseinate. Heat-treatment significantly increased stability but showed a negative effect on the activity and degree of hydrolysis. Lower degrees of hydrolysis resulted in significantly higher chelating activity, while the opposite was observed for radical scavenging activity. Combining peptide abundance with bioinformatic prediction, we identified several peptides that are likely linked to the observed differences in bulk emulsifying properties. The study highlights the prospects of applying proteomics and bioinformatics for hydrolysate characterization and in food protein science.     

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC₅₀ of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.     

Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme–substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.     

Two Novel Antioxidant Nonapeptides from Protein Hydrolysate of Skate (Raja porosa) Muscle

In the current study, the preparation conditions of neutrase hydrolysate (SMH) from skate (Raja porosa) muscle protein were optimized using orthogonal L₉(3)⁴ tests, and R values indicated that pH was the most important factor affecting HO· scavenging activity of SMH. Under the optimum conditions of pH 7.0, enzymolysis temperature 60 °C, enzyme/substrate ratio (E/S) 2%, and enzymolysis time 5 h, EC₅₀ of SMH on HO· was 2.14 ± 0.17 mg/mL. Using ultrafiltration, gel filtration chromatography, and RP-HPLC, two novel antioxidant nonapeptides (SP-A and SP-B) were isolated from SMH and their amino acid sequences were found to be APPTAYAQS (SP-A) and NWDMEKIWD (SP-B) with calculated molecular masses of 904.98 Da and 1236.38 Da, respectively. Both showed strong antioxidant activities. SP-A and SP-B exhibited good scavenging activities on HO· (EC₅₀ 0.390 and 0.176 mg/mL), DPPH· (EC₅₀ 0.614 and 0.289 mg/mL), and O₂⁻· (EC₅₀ 0.215 and 0.132 mg/mL) in a dose-dependent manner. SP-B was also effective against lipid peroxidation in the model system. The aromatic (2Trp), acidic (2Asp and Glu), and basic (Lys) amino acid residues within the sequences of SP-B might account for its pronounced antioxidant activity. The results of this study suggested that protein hydrolysate and peptides from skate muscle might be effective as food additives for retarding lipid peroxidation occurring in foodstuffs.     

ACE Inhibitory Peptide from Skin Collagen Hydrolysate of Takifugu bimaculatus as Potential for Protecting HUVECs Injury

Angiotensin-I-converting enzyme (ACE) is a crucial enzyme or receptor that catalyzes the generation of potent vasopressor angiotensin II (Ang II). ACE inhibitory peptides from fish showed effective ACE inhibitory activity. In this study, we reported an ACE inhibitory peptide from Takifugu bimaculatus (T. bimaculatus), which was obtained by molecular docking with acid-soluble collagen (ASC) hydrolysate of T. bimaculatus. The antihypertensive effects and potential mechanism were conducted using Ang-II-induced human umbilical vein endothelial cells (HUVECs) as a model. The results showed that FNLRMQ alleviated the viability and facilitated apoptosis of Ang-II-induced HUVECs. Further research suggested that FNLRMQ may protect Ang-II-induced endothelial injury by regulating Nrf2/HO-1 and PI3K/Akt/eNOS signaling pathways. This study, herein, reveals that collagen peptide FNLRMQ could be used as a potential candidate compound for antihypertensive treatment, and could provide scientific evidence for the high-value utilization of marine resources including T. bimaculatus.     

Natural dye extraction from waste barks of Turkish red pine (Pinus brutia Ten.) timber and eco-friendly natural dyeing of various textile fibers

In this study, a natural dye extraction was carried out to isolate dyestuff extract powder from the waste barks of Turkish red pine (Pinus brutia Ten.) timber which is not a common natural dyestuff source. The natural dyestuff powder obtained was applied to cotton, flax, wool, silk, tencel, polyamide and acrylic fibers accompanied by simultaneous application of alum and natural oak ash mordants. Color properties were investigated including rub-, light- and wash-fastness performance. Differently dyed fiber samples exhibited slightly different shades of beige, brown and brownish-yellow depending on the mordant used and fiber type. Alum mordanted samples exhibited better color properties. The highest (63.4) and the second highest (45.3) f(k) color yield values were observed for alum mordanted silk and wool samples, respectively. Dyed fabrics showed excellent wash fastness, very low staining performance, and moderate light- and rub-fastness.     

Gracilaria gracilis (Gracilariales,Rhodophyta) from Dakhla (Southern Moroccan Atlantic Coast) as Source of Agar: Content,Chemical Characteristics,and Gelling Properties

Agar is a sulfated polysaccharide extracted from certain marine red algae, and its gel properties depend on the seaweed source and extraction conditions. In the present study, the seaweed Gracilaria gracilis (Gracilariales, Rhodophyta) from Dakhla (Moroccan Atlantic Coast) was investigated for its agar content, structure, and gel properties. The agar yields of G. gracilis were 20.5% and 15.6% from alkaline pretreatment and native extraction, respectively. Agar with alkaline pretreatment showed a better gelling property supported by higher gel strength (377 g·cm⁻²), gelling (35.4 °C), and melting (82.1 °C) temperatures with a notable increase in 3,6-anhydro-galactose (11.85%) and decrease in sulphate (0.32%) contents. The sulfate falling subsequent to alkaline pretreatment was verified through FT-IR spectroscopy. The ¹³C NMR spectroscopy showed that alkaline-pretreated agar has a typical unsubstituted agar pattern. However, native agar had a partially methylated agarose structure. Overall, this study suggested the possibility of the exploitation of G. gracilis to produce a fine-quality agar. Yet, further investigation may need to determine the seasonal variability of this biopolymer according to the life cycle of G. gracilis.     

Screening of medium components by Plackett-Burman design for carotenoid production using date (Phoenix dactylifera) wastes

This work was conducted to use palm date wastes in the production of carotenoids. Three strains of Lactobacillus plantarum (QS1, QS2 and QS3) were isolated from food samples including bakery's yeast, olive fermentation and cheese. Strains were investigated for their structural recA gene by PCR. The recA gene was successfully amplified from all strains under study. The ability of isolated L. plantarum strains to produce carotenoids in MRS broth was investigated. QS3 isolate gave the highest ability for production of carotenoids. Statistical screening of media components for production of carotenoids by L. plantarum QS3 using date syrup as a source of sugar was carried out using Plackett-Burman design. Date syrup at 5% sugar concentration produced 16.21 mg/kg dry cell of carotenoids when used alone. Increase in carotenoids production was recovered (54.89 mg/kg dry cell) with supplementation of MRS medium with salts and organic nitrogen after optimization of pH and temperature using date syrup as a carbon source. Plackett-Burman design showed peptone, K₂HPO₄, sodium acetate and date juice as main components affecting carotenoids production.     

Applicability of Industrial Sisal Fiber Waste Derived Activated Carbon for the Adsorption of Volatile Organic Compounds (VOCs)

Agricultural waste produced by the industry is a huge threat for the global environment. Utilization of agricultural waste is necessary and there is an urgent need to develop new techniques to solve this important problem. The main objective of this research was to evaluate the applicability of the activated carbon (AC) derived from industrial sisal fiber waste as passive samplers in monitoring toluene by comparing them to industry standard wafer and granular activated carbon (GAC). Carbonization time and ball milling effect on sisal fiber derived AC sample adsorption properties were investigated. Toluene adsorption isotherms were used to predict toluene adsorption capacities. Surface characteristics including surface area and pore volume were used to determine the relationships between them and adsorption capacity. Even though prepared AC samples have mesoporous structure, commercial samples have microporous structure. Surface area from 1245 m²/g to 1297 m²/g and toluene adsorption capacity from 21.4 % to 26.6 % was improved by increasing carbonization time from 1 h to 3 h at 650 °C carbonization temperature and 94.4 ml/min flow rate. Conversely, ball milling technique had negative effect by decreasing the surface area (674 m²/g) and the adsorption capacity of toluene (12.27 %). It is concluded that industrial sisal fiber waste have great potential as a precursor of AC for application in passive monitoring against toluene, particularly the produced mesoporous AC with 3 h carbonization time performs higher adsorption capacity (26.6 %) than commercially available microporous passive sampler (24.1 %) and GAC (22.8 %).     

Combined Effects of UVR and Temperature on the Survival of Crab Larvae (Zoea I) from Patagonia: The Role of UV-Absorbing Compounds

The aim of our study was to assess the combined impact of UVR (280–400 nm) and temperature on the first larval stage (Zoea I) of three crab species from the Patagonian coast: Cyrtograpsus altimanus, C. angulatus, and Leucippa pentagona. We determined the survival response of newly hatched Zoea I after being exposed for 8–10 h under a solar simulator (Hönle SOL 1200) at 15 and 20 °C. There was no mortality due to Photosynthetic Active Radiation (PAR, 400–700 nm) or ultraviolet-A radiation (UV-A, 315–400 nm), and all the observed mortality was due to ultraviolet-B radiation (UV-B, 280–315 nm). The data of larval mortality relative to exposure time was best fit using a sigmoid curve. Based on this curve, a threshold (Th) and the lethal dose for 50% mortality (LD₅₀) were determined for each species. Based on the Th and LD₅₀, C. altimanus was found to be the most resistant species, while L. pentagona was found to be the most sensitive to UV-B. For both species of Cyrtograpsus, mortality was significantly lower at 20 °C than at 15 °C; however, no significant differences between the two temperature treatments were found in L. pentagona. Bioaccumulation of UV-absorbing compounds in the gonads and larvae of C. altimanus, and to a lesser extent in C. angulatus, might have contributed for counteracting the impact of UV-B. However, most of the resilience to UV-B observed with the increase in temperature might be due to an increase in metabolic activity caused by a repair mechanism mediated by enzymes.     

The Effect of Thermal and Ultrasonic Treatment on Amino Acid Composition, Radical Scavenging and Reducing Potential of Hydrolysates Obtained from Simulated Gastrointestinal Digestion of Cowpea Proteins

The effect of thermal and ultrasonic treatment of cowpea proteins (CP) on amino acid composition, radical scavenging and reducing potential of hydrolysates (CPH) obtained from in vitro simulated gastrointestinal digestion of CP was evaluated. Hydrolysis of native and treated CP with gastrointestinal pepsin and pancreatin yielded CPH that displayed antioxidant activities based on oxygen radical scavenging capacity (ORAC), ferric reducing antioxidant power (FRAP) and superoxide radical scavenging activity (SRSA). CPH derived from the treated CP yielded higher ORAC values than CPH from untreated proteins. However, lower significant FRAP and SRSA values were observed for these samples compared to untreated CPH (p?<?0.05). Amino acid analysis indicated that CP processing decreased total sulphur-containing amino acids in the hydrolysates, particularly cysteine. The amount of cysteine appeared to be positively related to FRAP and SRSA values of CPH samples, but not ORAC. The results indicated that thermal and ultrasonic processing of CP can reduce the radical scavenging and reducing potential of the enzymatic hydrolysates possibly due to the decreased amounts of cysteine. Since the hydrolysates were generated with gastrointestinal enzymes, it is possible that the resulting compounds are produced to exert some health functions during normal consumption of cowpea.     

The Bright Side of Gelatinous Blooms: Nutraceutical Value and Antioxidant Properties of Three Mediterranean Jellyfish (Scyphozoa)

Jellyfish are recorded with increasing frequency and magnitude in many coastal areas and several species display biological features comparable to the most popular Asiatic edible jellyfish. The biochemical and antioxidant properties of wild gelatinous biomasses, in terms of nutritional and nutraceutical values, are still largely unexplored. In this paper, three of the most abundant and commonly recorded jellyfish species (Aurelia sp.1, Cotylorhiza tuberculata and Rhizostoma pulmo) in the Mediterranean Sea were subject to investigation. A sequential enzymatic hydrolysis of jellyfish proteins was set up by pepsin and collagenase treatments of jellyfish samples after aqueous or hydroalcoholic protein extraction. The content and composition of proteins, amino acids, phenolics, and fatty acids of the three species were recorded and compared. Protein content (mainly represented by collagen) up to 40% of jellyfish dry weight were found in two of the three jellyfish species (C. tuberculata and R. pulmo), whereas the presence of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) was significantly higher in the zooxanthellate jellyfish C. tuberculata only. Remarkable antioxidant ability was also recorded from both proteinaceous and non proteinaceous extracts and the hydrolyzed protein fractions in all the three species. The abundance of collagen, peptides and other bioactive molecules make these Mediterranean gelatinous biomasses a largely untapped source of natural compounds of nutraceutical, cosmeceutical and pharmacological interest.