Side streams from wheat processing, such as the bran and gluten fractions, show great potential as a feedstock for the production of novel food ingredients and materials. In this study, we prepared hybrid polysaccharide-protein hydrogels via enzymatic crosslinking of wheat bran arabinoxylan and gluten fractions. Arabinoxylan was first isolated from wheat bran via subcritical water extraction, which preserved the covalently bound ferulic acid moieties to the arabinoxylan core amenable for laccase crosslinking. Gluten was fractionated into its main protein components (glutenin and gliadin) via treatment with aqueous ethanol. Hydrogels with different contents of arabinoxylan and gluten were prepared, demonstrating the integration of the protein fractions within the polysaccharide gel network. Increased addition of gluten led to gradually softer hydrogels, suggesting that the gluten fractions were not involved in the covalent crosslinking with the ferulic acid moieties to any noticeable level. Freeze-drying and regeneration of the hydrogels led to a 3-fold–10-fold increase in the storage and loss moduli, depending on the sample. Analysis of the structure of the hydrogels revealed that the addition of gluten upon enzymatic crosslinking impacted the physical interactions and crystallinity of the arabinoxylan populations, resulting in phase separation of the protein and polysaccharide components. This study demonstrates that tunable hydrogels can be prepared from cereal side streams, with potential as functional plant-based food hydrocolloids with improved nutritional properties, combining dietary fibre and protein components.

This study explores the cascade extraction of polyphenols and pectins from apple pomace and their potential application as food emulsifiers. The extraction of polyphenols was achieved by a simple method using ethanol and resulted in remarkable antioxidant activity. Pectic polysaccharides were sequentially extracted (5, 10 and 15 min) using subcritical water at two different temperatures (120 °C and 140 °C) and three different pH conditions (pH 3.0, 5.0 and 7.0). Acidic pH resulted in higher pectin yields when combined with higher temperature, whereas neutral conditions were less selective towards pectin. Pectin with different branching patterns could be isolated by tuning the process conditions. Linear homogalacturonan (HG) was preferentially isolated at milder conditions, whereas more branched rhamnogalacturonan (RG-I) populations required longer extraction times. Subcritical water enabled the extraction of pectins with different degrees of methoxylation (DM) depending on the extraction pH, which enabled the production of pectin acid gels with varying rheological properties. Low DM pectin was directly recovered from apple pomace at lower pH, eliminating the need for its conventional derivatization from high DM pectin. The emulsifying properties of two apple pomace pectin fractions with high and low methoxyl content were compared; the pectin with higher methoxyl content showed higher emulsifying capacity and stability. This study demonstrates that the relative abundance of the different pectic domains and the methoxylation of pectin can be controlled by subcritical water extraction, which has important implications for the valorization of pectic-rich agricultural wastes into valuable food ingredients.

Whole grain flours contain polysaccharides with techno-functional and nutritional properties which make them good candidates as natural texturisers in foods and beverages, thus reducing the use of highly refined ingredients. However, the use of plant components to develop complex fluids and soft materials, requires an enhanced un-derstanding of the relationship between their physicochemical and rheological properties. Here, we systemati-cally investigated the shear and extensional rheological properties of aqueous suspensions of whole grain rye and oat flours. Our results indicated that both types of suspensions (3.5 wt %) showed similar shear thinning behaviour (n = 0.4) however, oat suspensions presented higher viscosity and gel-like behaviour (G'>G") compared to rye. Additionally, the oat suspensions exhibited an apparent extensional viscosity, which was not present in rye suspensions. The rheological properties of the continuous and disperse phases, separated by centrifugation, were investigated before and after starch hydrolysis and protein removal. Our results indicate that the distinct behaviour of oat suspensions is mainly due to the molecular structure of starch in the liquid phase of i.e oat starch had a higher amylose/amylopectin ratio than rye. Whilst the presence of protein and cell wall polysaccharides in the solid phase contribute to the overall rheology of the suspensions. Furthermore, our results show that the systems do not follow the Cox-Merz rule, indicating that they behaved as suspensions of soft particles rather than macromolecules in solution. Aqueous suspensions of whole grain rye and oat flours showed rheological properties that could be of interest to design low-medium viscosity food and beverage products.

Biocatalytical upgrading of side streams from agricultural biomass into multifunctional materials constitutes a very attractive option to increase the circularity of food and material systems. We propose the design of radical scavenging hydrogels with mechanical integrity and protective effects against reactive oxygen species by enzymatic crosslinking of arabinoxylans (AX) with high ferulic acid content extracted from corn fibre using subcritical water. We have compared the influence of two enzymatic systems, laccase/O2 and peroxidase/H2O2, on the biochemical structure, multiscale assembly, physicochemical properties, and radical scavenging activity of the polysaccharide hydrogels. Peroxidase crosslinking results in instant hydrogel formation, whereas laccase shows slower crosslinking kinetics, resulting in a more elastic gel network. Characterization by size exclusion chromatography, small angle X-ray scattering, and microscopy revealed structural differences in the network organization of the hydrogels produced by the two enzymes. Laccase crosslinking leads to smaller polymeric aggregates, promoting their progressive organization in network clusters that impact the overall ultrastructure. Conversely, the fast crosslinking induced by peroxidase results in higher porosity and forms larger and potentially more heterogeneous aggregates, which seem to hinder their subsequent association in clusters. Both AX hydrogels exhibit adequate biocompatibility and protective effects against in vitro cellular oxidative stress compared to an alginate reference. This constitutes a proof of concept of the potential application of radical scavenging hydrogels from agricultural side streams for biomedical and nutritional applications in wound healing, cellular repair and targeted delivery.

Feruloylated arabinoxylan (FAX) from cereal brans has large potential to generate multifunctional materials with customized macromolecular and nanostructural architectures and techno-functional properties. Here we investigate the chemical and structural mechanisms of hydrogel formation of wheat bran FAX following enzymatic crosslinking by laccase and a subsequent regeneration procedure involving freeze-drying and resuspension of the crosslinked FAX in different pH buffers, using a battery of biochemical, rheological and physical techniques. The laccase crosslinking induced the conversion of ferulic acid units into a wide diversity of dimeric forms, leading to an increased molecular weight and a closer-packing of the FAX chains. The regeneration step resulted in a remarkable increase in the viscosity and viscoelasticity for all tested pH values. The amount of crystallinity of FAX increased by enzymatic crosslinking, it was however decreased by the regeneration step. The structural characterization revealed that enzymatic crosslinking, in addition to the formation of covalent crosslinks, influences the physical intermolecular interactions between adjacent FAX domains, and the regeneration forms larger clusters with higher dynamic moduli. Our results reveal that both chemical and physical mechanisms influence the network formation and multiscale assembly of wheat bran FAX hydrogels, thus modulating their rheological properties fundamental for their use in food and biomedical applications.

The use of macroalgae in food products is growing due to their techno-functionality and nutritional properties. In this context, an increased understanding of the rheological properties which are relevant for manufacturing and texture is needed. Here we investigated the impact of thermal and mechanical treatments, including high pressure homogenisation (HPH), on the polysaccharide composition, microstructure, and rheological properties of brown algae Laminaria digitata suspensions (5 wt %). Monosaccharide analysis and immunolabeling of alginate in combination with confocal laser scanning microscopy, revealed a sequential release of different polysaccharides as result of the applied shear. Results showed that thermal treatment (70 degrees C 1 h) and mild shear lead to suspensions of clusters of cells and release of fucoidan and laminarin into the liquid phase, conferring shear thinning properties to the suspensions. High pressure homogenisation was able to completely break the macroalgae cells, reducing particle size and releasing other soluble polysaccharides, in particular alginate, conferring gel properties (G'>G'') to the suspensions. This study contributes to the knowledge of how to design sustainable, innovative and nutritious liquid/semiliquid food products containing macroalgae biomass.

Cereal production generates large quantities of by-products every year, which still remain underutilized. The hemicellulose fractions from cereal by-products are anticipated to play an important role in tomorrow's sustainable and bio-based circular economy. This thesis addresses the valorization of hemicelluloses from cereal bran into films and hydrogels, starting from their isolation and expanding to the evaluation of material properties and potential use in future applications.

Initial isolation of arabinoxylans (AX) from wheat bran was achieved by a lab-scale subcritical water extraction (SWE) maintaining their functional groups (i.e. ferulic acid) and the effect of prior protein isolation on AX extraction was studied. The protein isolation resulted in a looser structure of wheat bran, which increased the polysaccharide yields in subsequent SWE. The polymeric structure and ferulic acid groups were preserved to a large extent after both protein isolation and SWE. The extracted AX fractions had considerable antioxidant activity, rendering them potential sources for further material development.

Further isolation of larger quantities of wheat bran AX was achieved by pilot scale SWE and alkaline extraction, resulting in feruloylated and non-feruloylated AX fractions, respectively. The film formation and properties of these AXs were investigated in comparison with a wheat endosperm AX. The three AX were also chemically modified by acetylation and applied in films. Higher purity, molecular weight, and degree of substitution of the AX extracts led to better thermal and mechanical properties of their films. The thermal stability of the films was significantly improved after chemical acetylation however, the mechanical performance and permeability properties did not change. Bound ferulic acid in feruloylated AX films was found to have considerably higher antioxidant activity than external incorporation of free ferulic acid.

Hydrogels were produced by enzymatic crosslinking of feruloylated AX from both wheat and corn bran, which show distinct molecular structure and ferulic acid content. For wheat bran AX, hydrogels were obtained by laccase crosslinking and the following regeneration process, and their biochemical and biophysical properties were studied. The rheological properties of feruloylated AX were enhanced by enzymatic crosslinking and further improved by the regeneration, proving that their mechanical strength can be modulated by chemical and physical adjustments. For corn bran AX, crosslinking was applied by laccase and peroxidase to compare the properties of the resulting hydrogels. Laccase formed a more elastic hydrogel network whereas peroxidase crosslinking resulted in hydrogels with larger covalent polymer networks. As a result of these differences between the two enzymes, the hydrogel obtained by peroxidase crosslinking contained larger aggregates with lower clustering strength. The crosslinking was followed by a cell application of the AX hydrogels, where their protective effect against chemically induced oxidative stress was demonstrated. Both corn bran AX hydrogels provided adequate scavenging against reactive oxygen species produced by human colon cells. It was shown that the gelation of wheat bran AX is governed by physical interactions between the xylan backbones of adjacent chains and interactions between larger scale aggregates in addition to the covalent crosslinks. The gelation mechanism of highly substituted corn bran AX was instead hypothesized to proceed by interactions between side chains together with covalent crosslinks. This thesis demonstrated that AX-based hydrogels could find potential use in food and biomedical applications. The outputs of this thesis will contribute to the bioeconomy and sustainable development by valorizing food side fractions into new high-value materials.

Spannmålsproduktion genererar varje år biprodukter i stora mängder som fortfarande är outnyttjade. Hemicellulosor från spannmålbiprodukter förväntas spela en viktig roll i framtidens hållbara och biobaserade cirkulära ekonomi. Denna avhandling omfattar valorisering av hemicellulosor från spannmålskli till filmer och hydrogeler, vilken startar med isolering och utvidgas till utvärdering av materialegenskaper och eventuell användning vid vidare applikationer.

Arabinoxylaner (AX) extraherades initialt genom att använda en subkritisk vattenextraktion (SWE) i laboratorieskala under vilken de funktionella grupperna (ferulsyra) behölls intakta och effekten av tidigare proteinisolering i AX extraktion studerades. Proteinisoleringen resulterade i en lösare struktur hos vetekliet, vilket ökade utbytet av polysackarid i efterföljande SWE. Polymerstrukturen och ferulsyra grupperna bibehölls i stor utsträckning efter både proteinisolering och SWE. De extraherade AX fraktionerna uppvisade avsevärd antioxidantisk aktivitet, vilket gör dem till potentiella källor för ytterligare materialutveckling.

Ytterligare isolering av vetekli AX i större mängder uppnåddes genom pilotskala SWE och alkalisk extraktion, vilket resulterade i feruloylerade och icke-feruloylerade AX-fraktioner. Filmbildningen och egenskaperna hos dessa AX undersöktes och jämföredes med en veteendosperm AX. De tre AX modifierades också kemiskt genom acetylering och applicerades i filmer. Högre renhet, molekylvikt ochsubstitutionsgrad hos AX extrakten ledde till bättre termiska och mekaniska egenskaper hos dessa filmer. Filmernas termiska stabilitet förbättrades avsevärt efter kemisk acetylering, men de mekaniska prestandaoch permeabilitetsegenskaperna förändrades inte. Bunden ferulsyra i AX filmerna uppvisade betydligt högre antioxidantisk aktivitet än extern införlivning av fri ferulsyra.

Hydrogeler producerades genom enzymatisk tvärbindning av feruloylerad AX från både vete och majskli, som båda uppvisar distinkt molekylstruktur och ferulsyrahalt. Hydrogeler producerades från vetekli AX genom lackas tvärbindning och den följande regenereringsprocessen samt deras biokemiska och biofysikaliska egenskaper studerades. De reologiska egenskaperna hos feruloylerad AX förstärktes genom enzymatisk tvärbindning och förbättrades vidare genom regenereringen, vilket bevisar att deras mekaniska hållfasthet kan moduleras genom kemiska och fysiska justeringar. För majskli AX applicerades tvärbindning med lackas och peroxidas för att jämföra egenskaperna hos de resulterande hydrogelerna. Lackas bildade ett mer elastisk hydrogelnätverk medan peroxidas tvärbindning resulterade i hydrogeler med större kovalenta polymernätverk. Som ett resultat av dessa skillnader mellan de två enzymerna uppvisade hydrogelerna från peroxidas tvärbindning större aggregat med lägre klusterstyrka. Tvärbindningen följdes av en cellapplicering av AX hydrogelerna, där deras skyddande effekt mot kemiskt inducerad oxidativ stress påvisades. Båda majskli AX hydrogelerna uppvisade god avlägsningsförmåga gemtemot reaktiva syreföreningar som produceras av humana kolonceller. Det visades att gelningen av vetekli AX styrs av fysikaliska interaktioner mellan xylan skelettet i angränsande AX kedjor och interaktioner mellan aggregat i större skala i tillägg till de kovalenta tvärbindningarna. Geleringsmekanismen för starkt substituerad majskli AX antogs istället gå vidare genom interaktioner mellan sidokedjor tillsammans kovalenta tvärbindningar. Denna avhandling visade att AX-baserade hydrogeler kan hitta potentiell användning inom livsmedel och biomedicinska tillämpningar. Resultatet av denna avhandling kommer att bidra till bioekonomi och hållbar utveckling genom att valorisera livsmedelsfraktioner till nya högvärdiga material.

Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.

This study demonstrates the potential of feruloylated arabinoxylan (AX) from wheat bran for the preparation of bioactive barrier films with antioxidant properties. We have comprehensively evaluated the influence of the structural features and chemical acetylation of feruloylated AX extracted by subcritical water on their film properties, in comparison with alkaline extracted AX and a reference wheat endosperm AX. The degree of substitution (DS) of AX had a large influence on film formation, higher DS yielded better thermal and mechanical properties. The barrier properties of AX films were significantly enhanced by external plasticization by sorbitol. Chemical acetylation significantly improved the thermal stability but not the mechanical or barrier properties of the films. The presence of bound ferulic acid in feruloylated AX films resulted in higher antioxidant activity compared to external addition of free ferulic acid, which demonstrates their potential use in active packaging applications for the preservation of oxygen-sensitive foodstuff. 

A cascade process for the sequential recovery of proteins and feruloylated arabinoxylan from wheat bran is proposed, involving a protein isolation step, enzymatic destarching and subcritical water extraction. The protein isolation step combining lactic acid fermentation and cold alkaline extraction reduced the recalcitrance of wheat bran, thus improving the total yields of the subsequent subcritical water extraction. The time evolution of subcritical water extraction of feruloylated arabinoxylan was compared at two temperatures (160 °C and 180 °C). Longer residence times enhanced the purity of target feruloylated arabinoxylans, whereas higher temperatures resulted in faster extraction at the expense of significant molar mass reduction. The radical scavenging activity of the extracted feruloylated arabinoxylans was preserved after the initial protein isolation step. This study opens new possibilities for the cascade valorization of wheat bran into enriched protein and non-starch polysaccharide fractions, which show potential to be used as functional food ingredients.