Exploring the bioactive potential of algae residue extract via subcritical water extraction: Insights into chemical composition and biological activity.

Gelidium sesquipedale is valued in the Spanish agar industry, but its production generates substantial waste, often discarded despite its nutritional and bioactive content. Subcritical water extraction (SWE) at 175 °C and 50 bar for 130 min was performed on this waste after agar extraction, comparing it to conventional ethanol extraction. The SWE extract exhibited superior nutritional profile, including proteins (170.6 ± 1.0 mg/gfreeze-dried-extract), essential amino acids (18.1%), carbohydrates (148.1 ± 0.3 mg/gfreeze-dried-extract), total phenolic content (57 ± 7 mg-EqGA/gfreeze-dried-extract), and also containing Maillard reaction compounds, such as 5-hydroxymethylfurfural, furfural, 2-furanmethanol, 1-(2-furanyl)-ethanone, and 5-methyl-2-furfural, influencing color, aroma and flavor. This extract showed better antioxidant and anti-inflammatory properties than the conventional extract, and higher xanthine oxidase, tyrosinase, and acetylcholinesterase inhibition activities. Toxicological assessment on human cells indicated the safety of the SWE extract. Therefore, SWE technology offers a promising method to valorize G. sesquipedale residue, yielding a bioactive and nutrient-rich extract suitable for food and nutraceutical applications.

Potential of Subcritical Water Hydrolysis to Valorize Low-Valued Ray-Finned Fish (Labeobarbus nedgia): Effects of Hydrolysis Temperature and Pressurization Agent.

Subcritical water (SCW) hydrolysis was applied to valorize the low-valued ray-finned fish (Labeobarbus nedgia) into valuable protein hydrolysates, employing N2 and CO2 as pressurization agents at varying temperatures (140, 160, 180, and 200 °C). The degree of hydrolysis (DH) and total free amino acid content increased with temperature for both pressurizing agents. The highest DH (54.5 ± 0.4%) and total free amino acid content (210 ± 1 mg/gprot) were observed at 200 °C when CO2 gas was used as the pressurizing agent. Predominantly, glycine and alanine were released for both pressurizing agents. The antioxidant activity, evaluated through three different assays, increased with temperature and was found to be the highest at 200 °C. This study illustrated the advantages of the intensified SCW technology by using CO2 as a pressurization agent in valorizing low-valued ray-finned fish (Labeobarbus nedgia), as animal residue rich in proteins, for the production of valuable protein hydrolysates with a high fraction of valuable free amino acids, which could offer potential applications as a functional ingredient in the food industry.

Green fractionation and hydrolysis of fish meal to improve their techno-functional properties.

A green strategy employing water as solvent has been adopted to obtain protein hydrolysates from fish meal (FM), its water-soluble fraction (WSP), and its non-water-soluble fraction (NSP). The techno-functional properties of the hydrolysates have been investigated and compared to hydrolysates obtained with Alcalase®. In general, SWH hydrolysates presented higher content of free amino acids and higher degree of hydrolysis, which reflected on the molecular size distribution. However, Alcalase® hydrolysates presented better solubility (from 74 ± 4% for NSP at pH = 2 up to 99 ± 1% for WSP at pH = 4-7). According to fluorescence experiments, FM and NSP hydrolysates showed the highest surface hydrophobicity, which has been related to better emulsifying properties and higher emulsion stability. The emulsions stabilized with 2%wt. of SWH-treated NSP showed the smallest particle sizes, with D[4,3] = 155 nm at day 0, and good stability, with D[4,3] = 220 nm at day 7, proving that water fractionation followed by SWH treatment is a good method to improve the techno-functional properties of the hydrolysates.

Onion (Allium cepa L.) Skin Waste Valorization: Unveiling the Phenolic Profile and Biological Potential for the Creation of Bioactive Agents through Subcritical Water Extraction.

Onion skin waste (OSW), the primary non-edible byproduct from onion processing, offers a renewable source of bioactive compounds. This study aims to valorize OSW through subcritical water extraction (SWE), aligning with a circular economy and biorefinery principles. SWE was carried out at 145 °C and 50 bar for 50 min in a discontinuous reactor, producing a phenolic-rich extract (32.3 ± 2.6 mg/g) dominated by protocatechuic acid (20.3 ± 2.5 mg/g), quercetin-4'-O-glucoside (7.5 ± 0.2 mg/g), and quercetin (3.2 ± 0.6 mg/g). Additionally, the extract contains sugars (207.1 ± 20.3 mg sucrose-Eq/g), proteins (22.8 ± 1.6 mg BSA-Eq/g), and free amino acids (20.4 ± 1.2 mg arginine-Eq/g). Its phenolic richness determines its scavenging activity against ●NO and O2●- radicals and its α-glucosidase and aldose-reductase inhibition without affecting α-amylase. Notably, the extract demonstrates significant α-glucosidase inhibition (IC50 = 75.6 ± 43.5 µg/mL), surpassing acarbose (IC50 = 129.5 ± 1.0 µg/mL) in both pure enzyme and cell culture tests without showing cytotoxicity to AGS, HepG2, and Caco-2 human cell lines. The extract's bioactivity and nutritional content make it suitable for developing antioxidant and antidiabetic nutraceutical/food components, highlighting SWE's potential for OSW valorization without using organic solvents.

Production of small peptides and low molecular weight amino acids by subcritical water from fish meal: Effect of pressurization agent.

The hydrolysis of the water-soluble protein (WSP) fraction from tuna fish meal was evaluated by subcritical water (subW) by using N2 and CO2 as different pressurization agents in the temperature range from 140 to 180 °C. For both gases, the amino group release increased by increasing working temperature while the Lowry response decreased due to production of smaller-size peptides and free amino acids. The free amino acid content was higher with CO2 than with N2. At 180 °C, 344 ± 5 and 275 ± 3 mg of free amino acids per g of WSP were released, respectively; although, in both systems the smallest molecular weight amino acids, glycine and alanine, were preferentially released. The free amino acids content obtained by enzymatic hydrolysis with commercial proteases Alcalase and Novozym was much lower with the highest hydrolysis yield determined for histidine. These results have been supported by size exclusion chromatography analysis.

Subcritical Water Treatment for Valorization of the Red Algae Residue after Agar Extraction: Scale-Up from Laboratory to Pilot Plant.

The feasibility of industrial subcritical water treatment on Gelidium sesquipedale residue through scaling up from the lab to pilot system in discontinuous mode (geometric scale-up factor = 50), at 130 and 175 °C (5% biomass), was investigated. The maximum volumes of the reactors were 500 mL at the lab-scale and 5 L at the pilot-scale system. At 175 °C, faster extraction/hydrolysis was observed for the pilot plant, but maximum yields were similar: 71.4 and 78.6% for galactans, 9.8 and 10.4% for glucans, and 92.7 and 86.1% for arabinans in pilot scale and lab scale, respectively, while the yields for proteins accounted nearly 40%. The highest yields for amino acids were observed for the smallest ones, while lower yields were determined for polar amino acids. The total phenolic content and color intensity progressively increased along time at lab scale, while a plateau was reached at the pilot level. Lower extraction yields but reproducible results were obtained at 130 °C. Finally, the pilot scale was essayed at a higher biomass loading (15%), and successful results were obtained, supporting the feasibility of the scaling-up process.

Subcritical Water as Pretreatment Technique for Bioethanol Production from Brewer's Spent Grain within a Biorefinery Concept.

Bioeconomy and environmental issues envisage industrial by-products such as Brewer's spent grain (BSG) as renewable resources for their recycling and reuse within a biorefinery concept. This study aimed to investigate the production of bioethanol from subcritical water (subW) pretreated BSG, following the conversion of the BSG biopolymers cellulose and hemicelluloses. The subW pretreatment was performed in a batch reactor at 174 °C, during 60 min and 5% (w/v) of dry BSG charge. The behavior of BSG biopolymers under subW pretreatment was monitored by evaluating the chemical composition of the liquid and solid streams and the chemical and structural changes caused in the solid residues by scanning electron microscope (SEM), CHNS elemental analysis and water retention value (WRV). The production of bioethanol from subW-pretreated BSG was assessed by separate hydrolysis and fermentation (SHF) and also by simultaneous saccharification and fermentation (SSF) by using the enzymatic cocktail Celluclast 1.5 L (40 FPU/gsolids) and the yeast Ethanol Red®. The higher bioethanol productivity (1.073 g∙L-1∙h-1) and concentration (32.18 g/L) were achieved by SSF with higher solids' loading (25%) and following a fed-batch strategy. These results suggest that subcritical water pretreatment is a promising technology for the valorization of BSG as a feedstock for second-generation bioethanol production.

Adjustable Gel Texture of Recovered Crude Agar Induced by Pressurized Hot Water Treatment of Gelidium sesquipedale Industry Waste Stream: An RSM Analysis.

A significant amount of bioactive compound-rich solid waste is released during the industrial phycocolloid-centric extraction of Gelidium sesquipedale. The impact of mild pressurized hot water extraction on repurposing this waste for the recovery of agar with an adjustable gel texture is investigated. A two-factor interaction response surface model assessed the influences of the operating temperatures (80 to 130 °C), times (45 and 150 min), pressures (1 to 70 bar), and algae concentrations (3 to 10% (w:v)). At a temperature of 100 °C, a pressure of 10.13 bar, a recovery time of 45 min, and a 10% algae concentration, the working parameters were considered ideal (w:v). Agar with a hardness of 431.6 g, an adhesiveness of -13.14 g.s-1, a springiness of 0.94, a cohesiveness of 0.63, and a gumminess of 274.46 g was produced under these conditions. A combined desirability of 0.78 was obtained for the exposed technology that retrieved gels with a minimum agar yield of 10% and thermal hysteresis between 39 ± 1 and 52 ± 0.5 °C. The fitted design can provide a high techno-commercial value to the agri-food industrial waste stream.

Valorization of brewer's spent grain by consecutive supercritical carbon dioxide extraction and enzymatic hydrolysis.

The double effect of supercritical carbon dioxide, sc-CO2, in a biorefinery concept applied to brewer's spent grain (BSG) was assessed in this work. Extraction conditions to remove and valorize the lipophilic fraction were studied (20-40 MPa and 40-80 °C) obtaining a maximum yield of 5.70 ± 0.07 g/100 gBSG at 80 °C and 40 MPa. High pressures and temperatures resulted in higher content of total phenolic and flavonoid compounds, as well as higher antioxidant capacity. It was observed an improvement of the enzymatic hydrolysis yield by cellulase in the sc-CO2 treated BSG compared to the non-treated. This improvement could be partially attributed to the removal of the lipid fraction and to morphological changes of BSG after sc-CO2. Based on this double benefit, sc-CO2 can play an important role on biomass valorization.

Preparation of Water-in-Oil Nanoemulsions Loaded with Phenolic-Rich Olive Cake Extract Using Response Surface Methodology Approach.

In this study, we aimed to prepare stable water-in-oil (W/O) nanoemulsions loaded with a phenolic-rich aqueous phase from olive cake extract by applying the response surface methodology and using two methods: rotor-stator mixing and ultrasonic homogenization. The optimal nanoemulsion formulation was 7.4% (w/w) of olive cake extract as the dispersed phase, and 11.2% (w/w) of a surfactant mixture of polyglycerol polyricinoleate (97%) and Tween 80 (3%) in Miglyol oil as the continuous phase. Optimum results were obtained by ultrasonication for 15 min at 20% amplitude, yielding W/O nanoemulsion droplets of 104.9 ± 6.7 nm in diameter and with a polydispersity index (PDI) of 0.156 ± 0.085. Furthermore, an optimal nanoemulsion with a droplet size of 105.8 ± 10.3 nm and a PDI of 0.255 ± 0.045 was prepared using a rotor-stator mixer for 10.1 min at 20,000 rpm. High levels of retention of antioxidant activity (90.2%) and phenolics (83.1-87.2%) were reached after 30 days of storage at room temperature. Both W/O nanoemulsions showed good physical stability during this storage period.

Subcritical Water Extraction of Phenolic Compounds from Onion Skin Wastes (Allium cepa cv. Horcal): Effect of Temperature and Solvent Properties.

The valorization of onion skin wastes (OSW) through the extraction, identification, and quantification of phenolic compounds was studied in this work, using subcritical water in a semicontinuous extractor (2.5 mL/min; 105-180 °C; 5 MPa). The extraction of flavonoids resulted to be fast (<30 min) and temperature sensitive (maximum at 145 °C; total flavonoids, 27.4 ± 0.9 mg/g dry OSW (DOSW)). The experimental results were fitted to the Weibull model. The influence of the solvent properties on the flavonoids quantification was found to be critical. A precipitate was formed once the extracts cooled down. If removed, a significant fraction of the high temperature extracted flavonoids (as much as 71%, at 180 °C) was lost. Such a condition affected especially those compounds that show extremely low solubility in water at room temperature, whereas quercetin glycosylated derivatives were less affected by the polarity change of the medium induced by the temperature change. It was demonstrated that it is necessary to re-dissolve the subcritical water extracts by the addition of ethanol, which led to a medium with a polarity equivalent to that obtained with water at high temperature. At 145 °C, quercetin (15.4 ± 0.4 mg/g DOSW) and quercetin-4'-glucoside (8.4 ± 0.1 mg/g DOSW) accounted for the 90% of the total flavonoids identified. By recovering high added value bioactive compounds from OSW the principles of circular economy were fulfilled, providing a new use for this agricultural waste.

Bioactive Compounds of a Wheat Bran Oily Extract Obtained with Supercritical Carbon Dioxide.

A wheat bran oily extract obtained with supercritical carbon dioxide at 25.0 ± 0.1 MPa and 40 ± 2 °C has been analyzed in order to determine some valuable bioactive compounds as alkylresorcinols, α-linolenic acid, steryl ferulates, tocopherols and phenolic compounds, which levels were around 47, 37, 18, 7 and 0.025 mg/g oily extract, respectively. To our knowledge, this is the first time that the presence of steryl ferulates has been observed in a supercritical fluid extract of wheat bran and that γ-tocopherol has been described in wheat bran oily extracts. Other common quality parameters, directly correlated with oxidative degradation, were also evaluated. Acidity values around 15% oleic acid were detected, while low levels of hydroperoxides (around 2.4 meq O2/kg) and very low levels of hexanal (0.21 ppb) were found. Composition of the wheat bran oily extract was stable during 155 days of storage at 21 °C and darkness, and only a slight decrease in alkylresorcinols and tocopherols contents (13% and 20%, respectively) was observed. These results indicated an attractive potential of the obtained oily extract for industrial applications as food ingredients, nutraceuticals, and others.

Water Ultrasound-Assisted Extraction of Polyphenol Compounds from Brewer's Spent Grain: Kinetic Study, Extract Characterization, and Concentration.

Brewer's spent grain (BSG) was chemically characterized obtaining 52.1% of carbohydrates, 17.8% protein, 5.9% lipids, 13.5% insoluble lignin and 24.3% of water-soluble extractives. This work has been focused on the study of polyphenol extraction of the extractive fraction by water ultrasound-assisted extraction. Selected extraction conditions were 47 °C and 21.7 mL water/gdry-BSG. The effect of solvent polarity on polyphenol extraction was studied by using ethanol aqueous mixtures, from 20% to 100% ethanol. The kinetics of polyphenol extraction have been fitted to the power law and the Weibull models yielding mean values of the root mean square deviation lower than 7.5%. Extracts have been characterized in terms of quantification of individual phenolic compounds by HPLC-DAD and protein and sugar soluble fractions (glucose, xylose, and arabinose). Polyphenol profile has been compared with other hydrolytic techniques, such as acid, basic and enzymatic hydrolysis, showing that ultrasound was not as effective as basic hydrolysis to release the phenolic acids esterified to the cell wall. A further centrifuge ultrafiltration concentration step was able to yield a retentate enriched in the protein fraction while individual phenolic compounds where mainly transferred to the permeate.

A comparative in vitro study of standard facemask jet nebulization and high-flow nebulization in bronchiolitis.

Aim of Study: The use of a nebulizer paired with high-flow nasal cannulas (HFNC) has been proposed for drug delivery in bronchiolitis. Particle size nebulized is a relevant factor determining the efficacy of the nebulization. We replicated in vitro the theoretical parameters most widely used in bronchiolitis and we compared the size of the droplet nebulized with a standard nebulizer and a nebulizer integrated into HFNC. Materials and Methods: We used laser diffraction to analyze the particle size nebulized (volume median diameter Dv50). The standard system was a jet nebulizer connected to a facemask with a flow rate of 8 L/min (JN). Three designs were used as nebulizers integrated into HFNC: a vibrating mesh nebulizer set 1) before (HFNC-BH) and 2) after (HFNC-AH) the humidifier, and 3) a jet nebulizer connected before the nasal cannula (HFNC-BNC). HFNC was used with neonatal (3-8 L/min) and infant cannulas (8-15 L/min). Results: Droplet size was similar among the three drugs studied. A lower particle size was obtained when using the nebulization system integrated into HFNC compared to the standard nebulizer, regardless of the flow rate and the nasal cannula used when the position of the nebulizer was before the nasal cannula (p < 0.05): 6.89 µm (JN), 2.49 µm (HFNC-BNC 3 L/min), 2.59 µm (HFNC-BNC 5 L/min), 2.44 µm (HFNC-BNC 8 L/min), 3.22 µm (HFNC-BNC 10 L/min), 3.23 µm (HFNC-BNC 13 L/min), 3.16 µm (HFNC-BNC 15 L/min). The particle size was lower in HFNC-BF compared to the HFNC-AH using neonatal nasal cannula (3-8 L/min) (p < 0.05). Conclusion: The use of a nebulizer integrated with HFNC has shown promising results in an experimental scenario of bronchiolitis. The particle size achieved with the nebulizer placed before the humidifier is equivalent to the one obtained via conventional nebulization, and it is even smaller when the integrated nebulizer is placed before the nasal cannulas.

Omega-3 encapsulation by PGSS-drying and conventional drying methods. Particle characterization and oxidative stability.

Particles from Gas-Saturated Solutions (PGSS)-drying has been used as a green alternative to encapsulate omega-3 polyunsaturated fatty acids (n-3 PUFAs) at mild, non-oxidative conditions. PGSS-dried particles have been compared to those obtained by conventional drying methods such as spray-drying and freeze-drying, finding encapsulation efficiencies (EE) up to 98% and spherical morphology for PGSS- and spray-dried particles. Freeze-dried powders showed irregular morphology and EE from 95.8 to 98.6%, depending on the freezing method. Differential scanning calorimetry (DSC) analysis revealed glass-transition and melting peaks of OSA-starch and a cold-crystallization peak corresponding to the encapsulated n-3 PUFA concentrate. Compared to conventionally dried powders, PGSS-dried microparticles showed lower primary and secondary oxidation after 28 days of storage at 4 °C. Ascorbic acid addition combined with the mild processing conditions of PGSS-drying yielded particles with a maximum peroxide value of 2.5 meq O2/kg oil after 28 days of storage at 4 °C.

Substrates emulsification process to improve lipase-catalyzed sardine oil glycerolysis in different systems. Evaluation of lipid oxidation of the reaction products.

Mono- and diacylglycerols rich in omega-3 have a great interest due to their good bioavailability and oxidation stability compared with other kind of omega-3 concentrates. The main drawback in mono- and diacylglycerols production by glycerolysis is the immiscibility of the substrates, oil and glycerol. To improve mass transfer rates, avoiding the use of organic solvents, emulsification of both reactants as reverse micelles (glycerol-in-oil) was carried out previous to lipase-catalyzed sardine oil glycerolysis. Substrate emulsification yielded higher reaction rates compared to kinetics with no previous emulsification, but still lower than in organic solvents. To avoid the use of organic solvent, SC-CO2 was used as reaction medium but no kinetic advantages were demonstrated in the pressure range from 15 to 25 MPa. By increasing temperature, from 40 to 90°C, reaction rates increased both in a solvent-free system and in SC-CO2 medium. It was also found that an increase in temperature does not lead to an increase in the final oxidation status of the reaction products. This behavior was due to the adsorption capacity of the Lipozyme 435 support, giving lower oxidation status at the highest temperature, 80-90°C.

Production and concentration of monoacylglycerols rich in omega-3 polyunsaturated fatty acids by enzymatic glycerolysis and molecular distillation.

Production of monoacylglycerols (MAGs) rich in ω-3 polyunsaturated fatty acids (n-3 PUFAs) was conducted through short path distillation (SPD) of an acylglycerol mixture (containing 67% MAGs) produced by enzymatic glycerolysis of sardine oil with glycerol. A stepwise SPD process in a UIC KDL 5 system (vacuum 10(-3)mbar, feeding flow 1.0 mL/min) was proceeded: the first distillation performed at evaporator temperature (TE) of 110 °C to remove glycerol completely and most of FFAs; and the second distillation at optimized TE 155 °C; resulting in a stream distillate with 91% purity and 94% overall recovery of MAGs. This work also demonstrated that SPD is able to concentrate n-3 PUFAs in MAG form by distilling at proper TE e.g. 125 °C, where n-3 PUFAs are concentrated in the residues. Moreover, this work mapped out a complete processing diagram for scalable production of n-3 PUFAs enriched MAGs as potential food emulsifier and ingredient.

Kinetic study for the ethanolysis of fish oil catalyzed by lipozyme(®) 435 in different reaction media.

The ethanolysis of fish oil in various reaction medium (tert-pentanol, n-hexane and solvent free system) catalyzed by the immobilized commercial lipase Lipozyme(®) 435 (Candida Antarctica) at atmospheric pressure has been studied in this work. The effect of some kinetic parameters, such as the amount of lipase, temperature and the initial reactant molar ratio ethanol:oil on monoacyglyceride and ethyl ester yield has been analyzed. Experimental data were successfully correlated by a simple kinetic model based on the elementary reactions proposed in this work. At high initial reactant molar ratio the three elementary steps can be considered as irreversible. However the reaction rate constants ratio for the deacylation of monoglyceride to glycerol decreased by decreasing the molar ratio ethanol:oil. The reaction rates are slower in n-hexane as reaction medium compared to tert-pentanol and a solvent-free system, at the experimental conditions essayed in this work. In this last case, ethanol acts as solvent for reaction and as reactant.

Formulation and characterisation of wheat bran oil-in-water nanoemulsions.

Wheat bran oil (WBO) has been reported to have an important content of bioactive compounds, such as tocopherols, alkylresorcinols, steryl ferulates and other phenolic compounds; however, its poor solubility in water systems restricts its applications in the food industry. This study is focussed on the formulation of oil-in-water (O/W) nanoemulsions of WBO in order to improve the bioaccessibility of its active compounds. The influences of oil concentration, surfactant type and concentration, and emulsification method, on the droplet size and stability of the nanoemulsions were investigated. Response surface methodology was used to optimise the conditions for preparing stable nanoemulsions with the minimum droplet size. The optimal nanoemulsion was obtained when 1% of WBO and 7.3% of a surfactant mixture of Span 80 (37.4%) and Tween 80 (62.6%) were emulsified in water by high intensity ultrasonication for 50s after pre-emulsification with a high speed blender during 5 min. The optimal nanoemulsion showed good stability over time and antioxidant and tyrosinase inhibitory activities, which make it suitable for use in food applications.

Characterization of triacylglycerol composition of fish oils by using chromatographic techniques.

Triacylglycerols (TAG) of two different refined fish oils from sardine and a mixture of tuna and sardine oil were separated by reverse phase high performance liquid chromatography (RP-HPLC) with a binary solvent gradient of acetone/acetonitrile. Different fractions were observed in the chromatogram and TAG species were tentatively identified by subsequent analysis of the fatty acid (FA) profile in each fraction by capillary Gas Chromatography (GC). Peak identities were assigned on the basis of a multiple linear regression analysis by using factors such as carbon number, number of double bonds, number of monounsaturated fatty acids (MUFA) and number of polyunsaturated fatty acids (PUFA) in the molecule as predictors for TAG retention time. A successful correlation was obtained between retention times and the equivalent carbon number (ECN) of triacylglycerols. Regiospecific analysis of fatty acids in the TAG has been conducted by ethanolysis of the fish oil by using an immobilized lipase. The subsequent separation of 2-monoacylglycerol (2-MAG) by TLC (thin layer chromatography) analysis showed that ethanolysis system is effective for analysis of FA composition at the 2-position in oils containing PUFA. Principal components analysis (PCA) has been also applied to establish correlations between the different fatty acids in the TAG.