Production and Physicochemical Characterization of Gelatin and Collagen Hydrolysates from Turbot Skin Waste Generated by Aquaculture Activities.

Rising trends in fish filleting are increasing the amount of processing by-products, such as skins of turbot, a flatfish of high commercial value. In line with circular economy principles, we propose the valorization of turbot skins through a two-step process: initial gelatin extraction described for the first time in turbot, followed by hydrolysis of the remaining solids to produce collagen hydrolysates. We assayed several methods for gelatin extraction, finding differences in gelatin properties depending on chemical treatment and temperature. Of all methods, the application of NaOH, sulfuric, and citric acids at 22 °C results in the highest gel strength (177 g), storage and loss moduli, and gel stability. We found no relation between mechanical properties and content of pyrrolidine amino acids, but the best performing gelatin displays higher structural integrity, with less than 30% of the material below 100 kDa. Collagen hydrolysis was more efficient with papain than alcalase, leading to a greater reduction in Mw of the hydrolysates, which contain a higher proportion of essential amino acids than gelatin and show high in vitro anti-hypertensive activity. These results highlight the suitability of turbot skin by-products as a source of gelatin and the potential of collagen hydrolysates as a functional food and feed ingredient.

Optimal Recovery of Valuable Biomaterials, Chondroitin Sulfate and Bioapatites, from Central Skeleton Wastes of Blue Shark.

The industrial filleting of blue shark (Prionace glauca) led to the generation of a large number of central skeletons of low interest to fishmeal plants handling such wastes. In this context, the present study describes the optimization of the hydrolysis process (pH 8.35, T 58 °C, 1% (v/w) of alcalase and t = 4 h) to produce chondroitin sulfate (CS) together with the recovery of bioapatites. Then, that hydrolysate was chemically treated with an optimal alkaline-hydroalcoholic-saline solution (0.48 M of NaOH, 1.07 volumes of EtOH and 2.5 g/L of NaCl) and finally purified by ultrafiltration-diafiltration (30 kDa) to obtain glycosaminoglycan with a purity of 97% and a productive yield of 2.8% (w/w of skeleton). The size of the biopolymer (CS) was of 58 kDa with prevalence of 6S-GalNAc sulfation (4S/6S ratio of 0.25), 12% of GlcA 2S-GalNAc 6S and 6% of non-sulfated disaccharides. Crude bioapatites were purified by pyrolysis and FT-Raman and XRD techniques confirm the presence of hydroxyapatite [Ca5(PO4)3(OH)], with a molar mass of 502.3 g/mol, embedded in the organic matrix of the skeleton. The mineralized tissues of blue shark are promising marine sources for the extraction of high value biomaterials with clinical application in bone and tissue regeneration and are still completely unexplored.

Chondroitin sulfate and hydroxyapatite from Prionace glauca shark jaw: Physicochemical and structural characterization.

In the present work, the potential of the Prionace glauca jaw as a source of both chondroitin sulfate and bioapatite is explored. The sandwich-type structure in cross section of the jaw based on alternate layers with prevalence in organic tissue or mineralized is shown and these bands respectively confirmed as CS or hydroxyapatite -enriched zones. As result of this, an optimized process in sequential steps for the recovery of both biomaterials and their purification process is proposed, by combining enzymatic proteolysis, chemical precipitation and separation using ultrafiltration membrane for CS production together with controlled thermal treatment for hydroxyapatite obtaining. The purified CS was characterized by Gel Permeation Chromatography, Nuclear Magnetic Resonance and Strong Anion Exchange Chromatography, revealing a polymeric material with a molecular weight of 67 kDa, and prevalent 6S-GalNAc sulfation (68%), followed by 4S-GalNAc (13%), a significant proportion of disulfated disaccharides (12%) and only 7% of non-sulfated units. In the case of the bioapatite a purified biphasic 60:40 porous calcium phosphate of hydroxyapatite: whitlockite/ß-TCP was confirmed. Hydroxyapatite as major component (85%) was also obtained for jaws directly subjected to the thermal treatment. This proved the influence of the enzymatic hydrolysis and centrifugation on the composition of the mineral fraction.

Optimal isolation and characterisation of chondroitin sulfate from rabbit fish (Chimaera monstrosa).

Chondroitin sulfate (CS) is a glycosaminoglycan widely explored for cartilage regeneration. Its bioactivity is influenced by sulfation degree and pattern, and distinct sulfation in marine CS may open new therapeutic possibilities. In this context, we studied for the first time the isolation and characterisation of CS from Rabbit Fish (Chimaera monstrosa). We propose an efficient process starting with enzymatic hydrolysis, followed by chemical treatments and ending in membrane purification. All steps were optimised by response surface methodology. Chemical treatment by alkaline-hydroalcoholic precipitation led to 99% purity CS suitable for biomedical and pharmaceutical applications, and treatment by alkaline hydrolysis yielded CS adequate for nutraceutical formulations (89% purity). Molecular weight and sulfation profiles were similar for both materials. Gel permeation chromatography analyses resulted in molecular weights (Mn) of 51-55 kDa. NMR and SAX-HPLC revealed dominant 6S-GalNAc sulfation (4S/6S ratio of 0.4), 17% of GlcA 2S-GalNAc 6S and minor quantities of other disaccharides.

Isolation and Chemical Characterization of Chondroitin Sulfate from Cartilage By-Products of Blackmouth Catshark (Galeus melastomus).

Chondroitin sulfate (CS) is a glycosaminoglycan actively researched for pharmaceutical, nutraceutical and tissue engineering applications. CS extracted from marine animals displays different features from common terrestrial sources, resulting in distinct properties, such as anti-viral and anti-metastatic. Therefore, exploration of undescribed marine species holds potential to expand the possibilities of currently-known CS. Accordingly, we have studied for the first time the production and characterization of CS from blackmouth catshark (Galeus melastomus), a shark species commonly discarded as by-catch. The process of CS purification consists of cartilage hydrolysis with alcalase, followed by two different chemical treatments and ending with membrane purification. All steps were optimized by response surface methodology. According to this, the best conditions for cartilage proteolysis were established at 52.9 °C and pH = 7.31. Subsequent purification by either alkaline treatment or hydroalcoholic alkaline precipitation yielded CS with purities of 81.2%, 82.3% and 97.4% respectively, after 30-kDa membrane separation. The molecular weight of CS obtained ranges 53⁻66 kDa, depending on the conditions. Sulfation profiles were similar for all materials, with dominant CS-C (GlcA-GalNAc6S) units (55%), followed by 23⁻24% of CS-A (GlcA-GalNAc4S), a substantial amount (15⁻16%) of CS-D (GlcA2S-GalNAc6S) and less than 7% of other disulfated and unsulfated disaccharides.

Determination of lactadherin concentration in dairy by-products by ELISA: Effect of heat treatment and hydrolysis.

Lactadherin is a peripheral glycoprotein of the milk fat globule membrane with several attributed biological activities. In this study, we developed an indirect competitive ELISA to determine lactadherin concentration by using a rabbit polyclonal antiserum. The ELISA was applied to quantify lactadherin in several dairy by-products. Of the products tested, raw and commercial buttermilk had the highest concentrations of lactadherin (6.79 and 5.27 mg/g of product, respectively), followed by commercial butter serum (4.86 mg/g), commercial skim milk (4.84 mg/g), and raw whey (1.20 mg/g). The concentration of immunoreactive lactadherin was also determined in dairy by-products after they were subjected to different technological treatments. Thus, raw products were heat treated at combinations of temperature and time typically used in the dairy industry, and commercial products were hydrolyzed using 3 proteolytic enzyme preparations. Heat treatments of whey and buttermilk resulted in a smaller decrease in lactadherin concentration than did hydrolysis as determined by ELISA and electrophoresis. At high temperatures for long durations, the loss of lactadherin was higher in whey than in buttermilk, with the maximal reduction of around 48% found after treating whey at 72°C for 60 min. Hydrolysis of commercial products with proteolytic enzymes resulted in a marked decrease of immunoreactivity within the first 5 min of treatment, which thereafter was constant throughout 4 h of hydrolysis. These results demonstrate that dairy by-products from milk fat processing are good natural sources of lactadherin, although technological processes have to be considered, because they have different effects on lactadherin content.

Optimisation of the extraction and purification of chondroitin sulphate from head by-products of Prionace glauca by environmental friendly processes.

The goal of the present work was to optimise the different environmental friendly processes involved in the extraction and purification of chondroitin sulphate (CS) from Prionace glauca head wastes. The experimental development was based on second order rotatable designs and evaluated by response surface methodology combined with a previous kinetic approach. The sequential stages optimised were: (1) the enzymatic hydrolysis of head cartilage catalysed by alcalase (55.7 °C/pH 8.2); (2) the chemical treatment of enzyme hydrolysates by means of alkaline-hydroalcoholic saline solutions (NaOH: 0.54 M, EtOH: 1.17 v, NaCl: 2.5%) to end the protein hydrolysis and to precipitate and selectively redissolve CS versus the peptidic material and (3) the selective purification and concentration of CS and the concomitant protein permeation of extracts which were obtained from previous treatment using ultrafiltration and diafiltration (UF-DF) technologies at two different cut-offs.

Production of Chondroitin Sulphate from Head, Skeleton and Fins of Scyliorhinus canicula By-Products by Combination of Enzymatic, Chemical Precipitation and Ultrafiltration Methodologies.

This study illustrates the optimisation of the experimental conditions of three sequential steps for chondroitin sulphate (CS) recovery from three cartilaginous materials of Scyliorhinus canicula by-products. Optimum conditions of temperature and pH were first obtained for alcalase proteolysis of head cartilage (58 °C/pH 8.5/0.1% (v/w)/10 h of hydrolysis). Then, similar optimal conditions were observed for skeletons and fin materials. Enzymatic hydrolysates were subsequently treated with a combination of alkaline hydroalcoholic saline solutions in order to improve the protein hydrolysis and the selective precipitation of CS. Ranges of 0.53-0.64 M (NaOH) and 1.14-1.20 volumes (EtOH) were the levels for optimal chemical treatment depending on the cartilage origin. Finally, selective purification and concentration of CS and protein elimination of samples obtained from chemical treatment, was assessed by a combination of ultrafiltration and diafiltration (UF-DF) techniques at 30 kDa.

Chondroitin sulfate, hyaluronic acid and chitin/chitosan production using marine waste sources: characteristics, applications and eco-friendly processes: a review.

In the last decade, an increasing number of glycosaminoglycans (GAGs), chitin and chitosan applications have been reported. Their commercial demands have been extended to different markets, such as cosmetics, medicine, biotechnology, food and textiles. Marine wastes from fisheries and aquaculture are susceptible sources for polymers but optimized processes for their recovery and production must be developed to satisfy such necessities. In the present work, we have reviewed different alternatives reported in the literature to produce and purify chondroitin sulfate (CS), hyaluronic acid (HA) and chitin/chitosan (CH/CHs) with the aim of proposing environmentally friendly processes by combination of various microbial, chemical, enzymatic and membranes strategies and technologies.

Hyaluronic acid production by Streptococcus zooepidemicus in marine by-products media from mussel processing wastewaters and tuna peptone viscera.

BACKGROUND: Hyaluronic acid is one of the biopolymers most commonly used by the pharmaceutical industry. Thus, there is an increasing number of recent works that deal with the production of microbial hyaluronic acid. Different properties and characteristics of the fermentation process have been extensively optimised; however, new carbon and protein sources obtained from by-products or cheap substrates have not yet been studied. RESULTS: Mussel processing wastewater (MPW) was used as a sugar source and tuna peptone (TP) from viscera residue as a protein substrate for the production of hyaluronic acid (HA), biomass and lactic acid (LA) by Streptococcus zooepidemicus in batch fermentation. Commercial medium formulated with glucose and tryptone was used as the control. The parametric estimations obtained from logistic equations and maintenance energy model utilized for modelling experimental data were compared in commercial and low-cost media. Complete residual media achieved high production (3.67, 2.46 and 30.83 g l(-1) of biomass, HA and LA respectively) and a high molecular weight of HA (approximately 2500 kDa). A simple economic analysis highlighted the potential viability of this marine media for reducing the production costs by more than 50%. CONCLUSIONS: The experimental data and mathematical descriptions reported in this article demonstrate the potential of media formulated with MPW and TP to be used as substrates for HA production by S. zooepidemicus. Furthermore, the proposed equations accurately simulated the experimental profiles and generated a set of interesting parameters that can be used to compare the different bacterial cultures. To the best of our knowledge, this is the first work in which a culture media formed by marine by-products has been successfully used for microbial HA production.