Subcritical Water Fractionation of bioactive compounds from Brewer’s Spent Grain (BGS)

Póster presentado en: 17th European Meeting on Supercritical Fluids. 2019, 8-11 de abril, Ciudad RealJCyL and ERDF for financial support of project BU301P18. To Hiperbaric, S.A. for financial support of Project BIOLIGNO

Semi-continuous hydrolysis of onion skin wastes with subcritical water: pectin recovery and oligomers identification

The semi-continuous hydrolysis of onion skin wastes (OSW) using subcritical water (SubW; working conditions: 105-180 ºC; 5 MPa; 2.5 mL/min) has been studied in this work. Liquids after hydrolysis showed a sharp increase in total organic carbon (>30 mg/g OSW) at temperatures above 145 ºC, when SubW began to promote the partial hydrolysis of the structural components of OSW. Among them, pectin was one of main components recovered (extraction yield up to 9% at 145 ºC), whereas cellulose was barely hydrolyzed in the range of temperatures studied. The composition of pectin demonstrated that SubW promoted the recovery of the valuable RG-I (21.1±1.1 mol %) regions compared to acid water extraction processes (10.8±0.9 mol %). The control of the hydrolysis conditions was found to be critical, since high temperatures and long hydrolysis times led to the formation of organic acids (acetic, formic and levulinic) and degradation products such as furfural (up to 0.8 mg/g OSW) from the C5 sugars. Finally, the High Heating Value (HHV) calculated for the solid residue obtained at 180 ºC (16.4±0.2 MJ/kg) indicated the potential use of this residue as a fuel, once the valuable compounds (phenolics and pectins) have been recovered.Agencia Estatal de Investigación (AEI) though projects PID2020-116716RJ-I00/ AEI / 10.13039/501100011033 and PID2019-104950RB-I00 / AEI / 10.13039/501100011033 and by the Junta de Castilla y León (JCyL) and the European Regional Development Fund (ERDF) through projects BU301P18 and BU050P20. Benito-Román post-doctoral contract was funded by JCyL and EDRF through project BU050P20 and by AEI through project PID2020-116716RJ-I00. E. Díaz de Cerio post-doctoral contract was funded by JCyL through project BU050P20. P. Alonso-Riaño predoctoral contract was funded by JCyL through ORDEN EDU/556/2019, de 5 de junio

Modification of brewer’s spent grain after sc-CO2 extraction: improvement of sugar and phenolic compounds release

Póster presentado en: EIFS2022, the 2nd Iberian Meeting on Supercritical Fluids (2º Encontro Ibérico de Fluidos Supercríticos / 2º Encuentro Ibérico de Fluidos Supercríticos), to be held on 28.February - 2.March 2022 in Coimbra, Portugal.Brewer ́s spent grain (BSG) is the solid by-product generated in breweries after the mashing and wort filtration process. It comprises about 85 % of the total by-products, generating approximately 20 kg per 100 L [1]. BSG presents a valuable chemical composition with a high content of protein and carbohydrates, 5 % of lipids and an important source of phenolic compounds. Among the different techniques proposed to valorize BSG, supercritical CO2 (sc-CO2) has become a promising technology to process biomass. The main aim of this work was to assess the effect of sc-CO2, after BSG oil extraction, on the subsequent enzymatic hydrolysis to hydrolyze the polysaccharide fraction into monomeric sugars by comparing the sugar yields of sc-CO2 and non-scCO2 treated BSG. After sc-CO2 treatment at 40 MPa and 80 oC, the raffinate obtained was subjected to enzymatic hydrolysis by cellulase at different enzyme dose. At the sc-CO2 extraction the carbohydrate fraction remained in the raffinate phase after extraction. Glucose yield increased with enzyme concentration for non-treated and sc-CO2 treated BSG. Higher yields of glucose were obtained for sc-CO2 treated compared to non-treated sc-CO2 for all the enzymes concentrations. The percentage increase in glucose yield for sc-CO2 treated and non-treated BSG was 8, 14 and 18 % for the three cellulase concentrations essayed in this work, 0.25, 0.5 and 1 %, respectively. The hydrolysis kinetics for xylose and arabinose have been also determined for non-treated and sc-CO2 treated BSG at 1 % of cellulase dose. Although not big differences were observed in the final sugar yield in the hydrolysates, the initial hydrolysis rate were significant higher for the sc-CO2 treated BSG than for the untreated samples. The higher enzymatic hydrolysis rate and yield obtained in the raffinate-BSG after sc- CO2 treatment compared with non-treated BSG could be attributed partially to the removal of the lipid fraction. As it has been described in literature [2], fats and oils could influence the susceptibility of carbohydrates to enzymes. This improvement was also due to surface morphology modification. The structural and chemical changes of untreated and sc-CO2 treated BSG was assessed by scanning electron microscopy and X-ray powder diffraction (XRD). Although the XRD pattern after sc-CO2 treatment indicated that the pre-treatment was not strong enough to modify the BSG crystallinity, the raffinate exhibited an irregular porosity and lamellar structure. sc-CO2 broke partially some structural barriers allowing a better enzyme access. Furthermore, some phenolic compounds were determined in the enzymatic hydrolysates at the end of the hydrolysis by using 1 % of cellulase (Table 1). It was observed that for p-coumaric acid and ferulic acid, a concentration 31 and 24 % higher, respectively, was obtained in the BSG hydrolysates after sc-CO2 treatment, while for vanillin similar concentration was obtained in both hydrolysates. The concentration of p-coumaric acid and vanillin after cellulase hydrolysis was lower than the values previously reported for the same BSG by alkaline, xylanase (1%) and subcritical water hydrolysis [3], [4]. Nevertheless, for ferulic acid, only a higher value was reached for alkaline hydrolysis.JCyL and ERDF for financial support of project BU050P20 Agencia Estatal de Investigación for financial support of project PID2019-104950RB-I00 / AEI / 10.13039/501100011033 JCyL and ESF for E. Trigueros (ORDEN EDU/574/2018) and P. Alonso-Riaño predoctoral (EDU/556/2019) contracts R. Melgosa is supported by a Beatriz Galindo Research Fellowship [BG20/00182

Kinetic study of the semi-continuous extraction/hydrolysis of the protein and polysaccharide fraction of the industrial solid residue from red macroalgae by subcritical water

The valorization of the underexploited solid residue after agar extraction from red marine algae was studied by subcritical water treatment. Experiments were carried out in two different semi-continuous fix-bed reactor configurations at 185 ºC at different subcritical water residence times. The use of a by-pass section allowed to heat the water previous contact to the biomass, avoiding the exposure of the sample to high temperatures during the heating procedure and reducing the formation of degradation products. Higher hydrolysis yields were obtained for the protein fraction (reaching 96.1%) than for the carbohydrate fraction (reaching 45.7%, 11.3%, 27.5% and 57.6% for galactans, glucans, arabinans and uronic acids, respectively). With the decrease of the residence time, by increasing the flow rate, higher initial hydrolysis rates were obtained due to enhancing diffusion of the hydrolysis products into the bulk solution. It was determined a similar dependence of the initial hydrolysis rates on the residence time for the carbohydrate oligomers and total protein fraction, but the release of free amino acids was less dependent on increasing flow rate due to higher diffusion coefficients for small molecules.Agencia Estatal de Investigación (Spain) [grant number PID2019–104950RB-I00 / AEI / 10.13039/501100011033], the Junta de Castilla y León (JCyL) and the European Regional Development Fund (ERDF) [grant numbers BU301P18 and BU050P20] E. Trigueros and P. Alonso-Riaño predoctoral contracts were funded by the JCyL and the European Social Fund (ESF) by ORDEN EDU/574/2018, de 28 de mayo and ORDEN EDU/556/2019, de 5 de junio, respectively. The C. I. K. Diop contract was funded by the European Commission, Horizon 2020 program through the Marie-Curie Individual Fellowship (H2020-MSCA-IF-2019), with regards to the ALGWAS-BIOR project (Grant agreement number 898804)

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.Junta de Castilla y León (JCyL) and ERDF for financial support of project BU301P18, and O. Benito-Román post-doctoral contract. To JCyL and ESF for the predoctoral contracts of E. Trigueros and P. Alonso-Riaño and for the contracts of D. Benito-Bedoya and D. M. Aymara-Caiza through the YEI program

Subcritical water fractionation of proteins and free amino acids from Brewer’s Spent Grain (BSG)

Póster presentado en: 1er Encuentro Ibérico de Fluidos Supercríticos/1º Encontro Ibérico de Fluidos Supercríticos. 2020, 18-19 de febrero, Santiago de CompostelaBrewer’s spent grain (BSG) has been traditionally used only in animal feed despite its high nutritional value, with a protein content of ~20% in dry weight basis [1]. This work is part of a wider project for a complete biomass valorization by using pressurized fluids, namely SC-CO2 to recover the lipophilic fraction and water. In this work we proposed the use of subcritical water at 50 bar and 4 ml/min in a semicontinuous reactor at different temperatures (125 to 185ºC) to extract and hydrolyze BSG proteins. Figure1 shows protein and amino acid values on SubCW extracts accumulated after 240 minutes of extraction. The highest protein content, 15.6 g/100g BSG, dry, was obtained at 185ºC. On the other hand, the highest free amino acid content in extracts, 77 μmol aa/g BSG, dry, was achieved at 160ºC due to amino acid decomposition at this operation conditions. This trend has been also observed in the literature for SCWF of oyster at 150 ºC [2]. Free amino acid formed during BSG protein hydrolysis in SubCW are listed in Table 1 together with the amino acid composition of the BSG protein fraction. Results appears to show a trend related to the hydrophobicity of each group of amino acid since an increase in temperature improves the yield of aliphatic amino acids extraction, while the highest yield for charged amino acids, was reached at 145ºC, and neutral amino acids with a polar side chain, had the highest yield at 160ºC. This agrees with the fact that aliphatic amino acids are stable at higher temperatures. The decrease of water polarity with temperature may favor the affinity for these amino acids due to their hydrophobic character. In addition, small aliphatic amino acids are formed during the decomposition of the other amino acids [3]. BSG offers a great potential as raw material to obtain protein hydrolyzates and amino acids due to its high protein content (~20%) and the elevated rate of EAA. Despite different amino acids has found to be a key parameter on the yield obtained for each amino acid as function of operating temperature. Results obtained in this work suggest that SCWF of BSG is able to recover and hydrolyze BSG proteins.JCyl and ERDF for financial support of project BU301P18. To Hiperbaric, S.A. for financial support of Project BIOLIGNO

Recovery of the protein fraction with high antioxidant activity from red seaweed industrial solid residue after agar extraction by subcritical water treatment

In this work valorization of the underexploited industrial solid residue generated after agar extraction from Gelidium sesquipedale was studied by using subcritical water in a semicontinuous fix-bed reactor. First, a complete characterization of this by-product was carried out, determining up to 21%(w/w) of protein content (for a nitrogen factor of 4.9) with high content of essential amino acids, 37% (w/w) of carbohydrate fraction and high amount of ash, 22% (w/w). The effect of temperature, in the range from 129 to 200 °C, and flow rate, in the range from 2 to 6 mL min−1, on protein and carbon fraction extraction/hydrolysiswas studied.At constant flow rate of 2 mL min−1, a maximum in the protein extraction was achieved at 185 °C. Higher temperatures led to degradation of protein or its hydrolysis products. Free amino acids release followed the same trend as the protein fraction. The most temperature sensitive amino acids, as determined by gas chromatography (EZ:faast Phenomenex), were serine and aspartic and glutamic acids. As a consequence, the selectivity towards non-polar amino acids increased by working at high severity factors. A Pearson correlation between antioxidant capacity of the collected extracts with the bioactive compounds determined in the extracts (total polyphenolic compounds –TPC-, peptides and free amino acids) was established, being stronger for TPC. The ash content in the solid residue after treatment steadily increased with temperature due to non-solubilization being possible its application in agriculture as fertilizers.Agencia Estatal de Investigación [grant number PID2019-104950RB-I00 / AEI / https://doi.org/10.13039/501100011033] and the Junta de Castilla y León (JCyL) and the European Regional Development Fund (ERDF) [grant number BU301P18 and BU050P20

Recovery of proteins and free amino acids from Gelidium Sesquipedale alga residue by subcritical water extraction (SWE)

Trabajo presentado en: 1er Encuentro Ibérico de Fluidos Supercríticos/1º Encontro Ibérico de Fluidos Supercríticos. 2020, 18-19 de febrero, Santiago de CompostelaGelidium sesquipedale is a red alga that provides the best raw material to obtain the highest quality agar in the spanish agar industry [1]. Industrial process generates a residue that is usually discarded; however, this by-product still contains important amounts of different valuable compounds, such as structural carbohydrates, bioactive compounds and proteins. To valorize by-product supplied by Hispanagar company (Burgos), subcritical water extraction (SWE) is a promising green technology since water presents unique properties as solvent [2]. This work is focused on the valorization of the protein fraction to produce free amino acids and to extract and hydrolyze protein into small peptides. A semi-continuous reactor has been used to perform the extraction, and protein fraction hydrolysis was studied at different temperatures (125, 140, 155, 170, 185, 200ºC), times, and solvent flow rate (2 and 6 mL/min). Total protein content in alga residue was 20.11±1.53% in which it was determined free amino acids, among which VAL, LEU, ILE, PHE, LYS and HIS are majority (Table 1), being total essential amino acids found 10.50±0.16%, whereas non-essential amino acids like GLY, PRO and GLU+GLN represent 4.57±0.06% Protein extraction grows with increasing temperature at constant flow, reaching a maximum at 200ºC. Moreover, when flow and temperature are increased, protein recovery shows the highest and fastest extraction because of its less residence time, what makes able to get a marked extraction yield improvement (Fig. 1). Amino acids extraction follows a similar trend than proteins: an increasing extraction up to a maximum at 185ºC when flow rate is 2 ml/min, but lower than 6 ml/min. Greatest extraction was found for ALA, GLY, SER and the mixture of ASN+ASP; also for MET, TYR and GLU+GLN mixture, with lower but remarkable yield (Table 1). At constant flow rate, maximum extraction is reached at 185ºC for all amino acids determined, decreasing at 200ºC. Nevertheless, flow rate increasing makes 200ºC extraction much higher, about all for GLU+GLN mixture, LYS, HIS, TYR, PRO, LEU and MET (Fig. 2). This agrees with other studies carried out with fish protein and amino acids standard [3,4]. Moreover, it has been noted than amino acids extraction presents a similar behaviour according to its properties: basic amino acids extraction remains practically constant, whereas than neutral and acid, hydrophobic and sulfur amino acids extraction increase when temperature raises. SWE is a useful technique to extract bioactive compounds. Parameters as temperature or solvent flow rate have much influence on the protein and amino acids extraction yield. All of this makes SWE an interesting alternative to conventional treatments.JCyL and ERDF for financial support of project BU301P18. To Hiperbaric, S.A. for financial support of Project BIOLIGNO. To JCyL and ESF for the predoctoral contracts of E. Trigueros and P. Alonso-Riaño an

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, scCO2 can play an important role on biomass valorization.This work was supported by the Agencia Estatal de Investigación [grant number PID2019-104950RB-I00 / AEI / https://doi.org/10.130 39/501100011033] and the Junta de Castilla y León (JCyL) and the European Regional Development Fund (ERDF) [grant numbers BU301P18 and BU050P20]. E. Trigueros and P. Alonso-Riano ˜ predoctoral contracts were funded by JCyL and the European Social Fund (ESF) by ORDEN EDU/574/2018, de 28 de mayo and ORDEN EDU/556/2019, de 5 de junio, respectively

Valorization of brewer’s spent grain by furfural recovery/removal from subcritical water hydrolysates by pervaporation

This work is focused on the development of a sustainable process for the valorisation of the main by-product generated in the brewing industry, the brewer’s spent grain (BSG). A two-step process combining subcritical water treatment and pervaporation (PV) was proposed to hydrolyse the hemicelluloses fraction of this lignocellulosic biomass and further removal/recovery of some of the degradation products of sugars by using two different organophilic membranes, polydimethylsiloxane (PDMS) and polyoctilmethylsiloxane (POMS) membranes. Specifically, furfural is the dehydration product of pentoses and it is one of the top biomass-based chemicals being an important platform chemical. For synthetic binary mixtures, lower total permeation flux but higher enrichment factors for furfural were determined for POMS. When dealing with subW hydrolysates, POMS membranes yielded the highest furfural recovery, 94.1 %, with permeate concentrations as high as 40 g⋅L1 . Furthermore, it was assessed that PV is a suitable detoxification method that yielded a retentate nearly free of furfural allowing its use as growth media in the opposite to the subW hydrolysate with inhibitory furfural concentrations for microbial bioprocesses.publishe