Plus de 40% des matériaux plastiques sont utilisés pour l’emballage. Les principaux polymères sur le marché de l’emballage alimentaire sont les polymères synthétiques d’origine fossile. Du fait de la raréfaction des ressources fossiles, la valorisation des polymères issus de matières premières renouvelables d’origine végétale représente une alternative à l’utilisation des polymères pétro-sourcés. Les hémicelluloses, avec la cellulose et les lignines, sont un composant majeur des ressources lignocellulosiques. Les hémicelluloses constituent des réseaux macromoléculaires denses et peu mobiles qui leur confèrent intrinsèquement d’excellentes propriétés barrières aux gaz. L’objectif de cette thèse est d’isoler les arabinoxylanes (AX) à partir de divers co-produits de céréales (son de blé, paille de blé, rafles de maïs) afin d’étudier l’impact de leurs compositions et de leurs structures sur certaines de leurs propriétés physico-chimiques, après mise en oeuvre sous forme de films pour des applications potentielles dans le domaine de l’emballage alimentaire.Les arabinoxylanes ont été extraits par un procédé alcalin puis séparés en trois fractions en fonction de leur solubilité dans l’eau et dans l’éthanol 50% (v/v). Les trois fractions récupérées (i/ insoluble dans l’eau, ii/soluble dans l’eau et insoluble dans l’éthanol, iii/soluble dans l’eau et soluble dans l’éthanol) présentent un rapport Ara/Xyl croissant avec leur solubilité dans l’eau et dans l’éthanol. Dans le cas du son de blé, les trois fractions présentent des masses moléculaires similaires mais se distinguent par différents degrés de substitution et divers types de substitutions de la chaîne principale de xylanes. L’étude des propriétés physico-chimiques de films formés par ces trois fractions ont permis d’établir une corrélation entre le degré de substitution et leurs propriétés sous forme de films. La morphologie cristalline, la mobilité des chaînes etl’hydratation des films de xylanes sont corrélés avec les taux de substitution des AX. Lorsque le taux d’Ara/Xyl diminue, le film est plus cristallin, la mobilité locale augmente alors que la mobilité globale diminue, la teneur en eau diminue à haute humidité relative (de 67% à 60% à 90%HR), la perméabilité et la diffusivité à l’oxygène diminue. L’étude des trois fractions insolubles dans l’eau isolée à partir de son de blé,de paille de blé et de rafles de maïs a révélé que les propriétés des films sont également affectées par la longueur des chaînes de xylanes et par leur conformation moléculaire ainsi que par la teneur des substituants tels que les acides uroniques. Des nanocharges (nanocristaux de cellulose de linters de coton) sont utilisées(5 ou 10% p/p) pour renforcer les films formés par les fractions d’AX de son de blé. L’incorporation des nanocristaux au sein des films n’a pas provoqué de modification du degré de cristallinité. Par contre, la teneur en eau à haute humidité relative est diminuée. La mobilité locale des chaînes d’AX est réduite indiquant l’existence d’interactions entre les nanocristaux et les AX alors que la mobilité globale des chaînes n’est pas modifiée. L’effet du renforcement semble être plus intense pour la fraction AX la plus substituée par les groupements arabinosyl. / There are more than 40% of plastics used for packaging. The main polymers in the food packaging market are synthetic polymers from fossil resource. Due to limited fossil resources on our planet, the development of renewable raw materials derived from plant is required to have an alternative to synthesized polymers from petroleum sources. Hemicelluloses, with cellulose and lignin, are a majorc omponent of lignocellulosic resources which display dense and low-mobility macromolecular networks making them have excellent gas barrier properties. The aim of this thesis is to isolate arabinoxylans (AX)from the various cereals co-products such as wheat bran, wheat straw and corn cob, in order to study the impact of their compositions and structures on several physicochemical properties of AX based films for potential food packaging application. The alkaline solution is used to extract arabinoxylans, and a step of fractionation is used to separate the different populations AX according to their solubility in water and in ethanol 50% (v/v). Three fractions were finally obtained (i) water insoluble, ii) water soluble-ethanol insoluble and iii) water ethanol soluble) displaying the increasing ratios Ara/Xyl with increasing solubility in water and in ethanol. The three fractions of wheat bran have similar molar masses, different substitution degrees and patterns. The results about the films physicochemical properties based on theset hree fractions could be established a relationship between the substitution degree and their films properties. The film morphology, the chain mobility, the water sorption behavior at high relative humidity and the coefficient permeability and diffusivity of oxygen could be correlated with the degree of substitution. When the Ara/Xyl ratio decreases, the film is more crystalline, the local chain mobilityincreases whereas the global chain mobility decreases, water content at high relative humidity decreases and the oxygen permeability and diffusivity decreases. The AX has different substitution patterns and molar masses according to their botanical origins. The study of three water-insoluble fractions isolated from three cereal co-products reveals that the film properties could be also affected by chain length, theamounts of other substituents such as uronic acids and their molecular conformations. The nanofillers(cellulose nanocrystals of cotton linters) are used in order to enhance the films properties based on threeAX fractions of wheat bran (5 or 10% w/w). The incorporation of nanocrystals in the films does not change in the crystalinity degree; whereas the water content at high relative humidity is decreased; the local chain mobility of AX is reduced indicating the existence of interactions between the nanocrystals and AX while the global chain mobility is almost unchanged. The reinforcement effect seems to be more intense for the most substituted AX by arabinosyl groups.

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.

Arabinoxylans (AX’s) are the predominant non-starch polysaccharides found in the structural matrix of cell walls in wheat grains, being present in large quantities in wheat bran, accounting for up to 25% of its composition. Their physicochemical properties define their functionality which can be beneficial in cereal-based products such as bread, where their addition could enhance the gluten matrix responsible for the aerated structure and quality of bread. Bioethanol production has grown rapidly, however, to be economically viable, cereal-derived first generation biorefineries need to adopt the process integration approaches employed in petrochemical refineries, and exploit the interaction opportunities arising from multiple product streams. A potential source of AX is its extraction from the wheat bran based low value animal feed produced as an end product of the bioethanol distillation process. The benefits of extraction are twofold, to enhance feed nutritional value by reducing fibre content and produce a high value product for use as a functional ingredient in the breadmaking industry. Extraction of AX involves precipitation with ethanol, giving opportunity for integration and economic extraction in the context of a bioethanol plant. Currently no commercial supply of AX is available in sufficient quantities to conduct functionality trials; therefore the objective of the current work was to study the feasibility of extracting AX from animal feed and, by developing a scaled-up extraction process based on that of Hollmann and Lindhauer (2005) and Du et al. (2009), to produce sufficient quantities to conduct functionality trials into its effects on the breadmaking process. Two animal feeds, representative of contrasting biorefinery operations, were used for AX extraction, Distillers’ Dried Grains with Solubles (DDGS) and C*Traffordgold®, with water and alkaline AX being extracted from each. Monosaccharide analysis of the feedstocks confirmed the presence of arabinoxylans, with DDGS containing 12.5% AX and TG 13% AX, with A/X ratios of 0.55 and 0.61, respectively. The purity of AX extracted at both lab scale and on scale up was consistent, with 29.5% and 23% extracted at lab scale and 30% and 25% extracted in the scaled up process for WEAX from DDGS and TG, respectively. The purity of AEAX was lower, with 18% and 14% at lab scale and 15% and 14% for scale up for DDGS and TG, respectively. The results indicate that the same purity of crude extract can be achieved at both lab and larger scale extraction, however lower yields and absolute yields were observed at scale up and anticipated to be due to the crude nature of the process, which needs further optimisation. Addition of crude AX extracts to bread dough gave insights into its effects and potential benefits and issues. The type and origin of AX was found to affect bread dough characteristics in different ways. WEAX from both DDGS and TG exhibited effects on all stages of the breadmaking process, by improving stability and reducing softening at mixing, and slowing proving allowing the dough to retain a better aerated structure, leading to an open aerated finished loaf structure. AEAX from both types of animal feed generally caused destabilization of gas cells and coalescence, resulting in loaves with a tighter crumb structure. The AX dosage appeared to have a non-linear effect, with some positive changes at 1% addition turning to negative changes when added at 2%. This work has provided insights into the functionality of AX in bread dough, and a process has been developed to allow greater than lab scale production of arabinoxylan. The effects of AX need to be understood more clearly to retain beneficial effects, whilst supressing detrimental ones, preferably while avoiding the need for extensive purification. The work supports the possibility for AX extracts to be produced commercially as bread ingredients that could enhance bread structure and nutritional quality, and provides an elegant solution for synergy between two wheat-based industries serving both the food and non-food needs of society.

This diploma thesis is focused on the use of wheat bran as a raw material for extraction of arabinoxylans. Wheat bran is a waste product of mill processing of wheat into flour and is mainly used as a livestock feed. However, the amount of bran produced significantly exceeds its consumption as feed, which opens up space for research into their further use. Polysaccharides such as cellulose, hemicelluloses and lignin are mainly present in the bran. The most common type of hemicelluloses are arabinoxylans, which form the main structural component of wheat bran. Alkaline extraction disrupts hydrogen and covalent bonds and releases polysaccharides of various molar weights from the crosslinked cell wall structure. Hydrolysis is required to obtain arabinose and xylose. Trifluoroacetic acid was chosen for the hydrolysis. The acid breaks down glycosidic bonds of the poly- and oligosaccharides to give the corresponding monosaccharides. The experiments made during this thesis have shown that the yield of multi stage extraction is twice as high as the yield of single stage extraction. Hydrolysis of the solid fractions after alkaline extraction revealed that a relatively large amount of arabinoxylans and other carbohydrates still remain bound in the complex matrix of the bran cell wall. Thus, alkaline extraction alone may not be the best option for obtaining arabinoxylans on a larger scale, or process optimization must be considered. Extraction with the addition of hydrogen peroxide appears to be effective for large-scale extraction processes.

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The content of beta-glucans and arabinoxylans in barley grain, malt and wort was monitored. The varieties of barley were Pionier, Libuše, Manta, Tango, KWS Amadora, Kampa, KWS Irina, Francin, Odyssey, Overture, Vendela, Petrus, Laudis 550, Sunshine, Kangoo, Xanadu, Sebastian, Bojos and Malz. These varieties were grown on three different habitats (Uherský Ostroh, Věrovany and Vysoká u Příbramě). The content of beta-glucans and arabinoxylans was also analysed in five different types of beer. In brewing and malting industry the content of beta-glucans and arabinoxylans is mainly monitored because of their negative influence on decanting of wort and filtration of beer. These substances can contribute to turbidity and precipitation in beer and thus negatively effect the stability of beer. The content of beta-glucans in barley grain, malt, wort and beer was determined by FIA method and the content of arabinoxylans was determined in barley grain, wort and beer by Douglas method. The highest content of beta-glucans in barley grain was found in 14/19 varieties at Uherský Ostroh, where the amount of beta-glucan content was in the range of 3,65-5,60 %. The highest beta-glucan content in malt was found in 17/19 varieties at Vysoká u Příbramě, where the beta-glucan content was between 0,15-0,79 % and the highest content of beta-glucans in wort was found in 16/19 varieties also at Vysoká u Příbramě. There was the beta-content in the range of 22-184 mg/l. The results of content of arabinoxylans in barley grain and wort were more balanced. The highest content of arabinoxylans in barley grain was found in 10/19 varieties at Věrovany. The content of arabinoxylans at this habitat was in the range of 3,04-4,56 %. The highest content of arabinoxylans in wort was determined in 8/19 varieties at Uherský Ostroh. The content of arabinoxylans at this habitat was in the range of 542-1040 mg/l. The highest content of beta-glucans in samples of beer was determined in a sample of light lager (207 mg/l) and the highest content of arabinoxylans was found in a samle of wheat lager (1465 mg/l).

Intensive and semi-intensive aquaculture systems are dependent on nutrient input either in the form of supplemental or complete feeds. Most complete diets still include high fish meal (FM) levels (≥10%). However, as the industry attempts to reduce its reliance on FM, feeds must now be formulated with much lower levels especially for omnivorous species such as tilapia. By 2015, mean FM inclusion in tilapia diets was projected to fall below 3% and be further reduced to 1% by 2020. In the global context of competition for crops, finding suitable plant-based replacers for FM and meeting the increasing demand for seafood, lower-cost and under-utilised plant feedstuffs are now receiving greater attention. The study was divided into three distinct components – field survey, growth experiments, and life cycle assessment. Field surveys were used to contextualise the growth experiments and assess commercialisation opportunities for multi-enzyme inclusion in tilapia feeds. Two sets of digestibility and growth experiments were designed to evaluate the feasibility of using high inclusions of plant-based ingredients sourced from locally available feedstuffs in Thailand to substitute FM at low inclusion levels (0 – 5%). The research evaluated the hypothesis regarding the potential of exogenous enzymes (protease, xylanase and phytase) to minimize anti-nutritional effects on nutrient digestibility of proteins, polysaccharides and phosphorus in tilapia. The research also assessed the secondary effects of enzyme supplementation on economic efficiency and life cycle environmental impacts. Tilapia is the second most cultured finfish globally and Thailand is the sixth largest producer. Based on the findings of the field survey, feeding practices of Thai tilapia farmers were confirmed to be diverse. Feed inputs included, but were not limited to, agro-industrial by-products (e.g. rice bran, corn bran etc.) and commercial diets. Commercial diets contained 15 – 30% crude protein and lower protein livestock diets (i.e. pig ration) were often used for supplemental feeding or “fattening”. The experimental low FM diets were therefore formulated as grow-out or “fattening” diets for semi-intensive green-water systems, a prominent feature (>60%) of Thai tilapia farming. In Phase 1, the digestibility experiment assessed the digestibility and growth in tilapia fed 0%, 3% and 5% FM diets with and without xylanase (0.385 g kg-1) and phytase (0.075 g kg-1). Performance decreased significantly with declining FM levels. No differences in feed intake, feed conversion ratio (FCR), specific growth rate (SGR) and weight gain were observed between the enzyme and control diets. Nevertheless, tilapia fed the enzyme supplemented 3% FM and control 5% FM performed similarly (P < 0.05). No enzyme-related effects were noticed for protein digestibility but phosphorus (P) digestibility improved by 9%, except at 0% FM level (P > 0.05). The enzymes had no apparent influence on nitrogen (N) retention contrary to previous studies, however, higher retention for P was observed. Villus length decreased with declining FM levels yet no improvements were seen in tilapia fed enzyme diets. In a simultaneous grow-out experiment, the six experimental diets were compared to an industry 10% FM standard. Conversion ratio was the lowest (1.66) in adult tilapia fed 10% FM diet however the enzyme supplemented 0% FM fed fish had a comparatively low FCR of 1.67. There were no significant enzyme-related effects on weight gain¸ SGR and protein efficiency. Proximal villi results were inconsistent. The cost of feed decreased with declining FM levels but increased with enzyme inclusion. Nevertheless, the economic returns per kg of whole fish produced were better using enzyme supplemented diets compared to the controls. Though the size of the effects on growth and nutrient utilisation were modest, the findings suggested that xylanase and phytase had some level of synergistic action on the targeted anti-nutrients. However, further research was required. In Phase 2, two control diets (2% FM, negative control (NC) and 10% FM, positive control (PC)) were compared with three enzyme supplemented 2% FM diets (NO-PRO, 0.385 g kg-1 xylanase and 0.075 g kg-1 phytase only; LO-PRO, xylanase + phytase + 0.2 g kg-1 protease and HI-PRO, xylanase + phytase + 0.4 g kg-1 PRO). Growth performances improved with enzyme supplementation compared to the NC (P < 0.05). Of the enzyme supplemented diets, the LO-PRO diet showed the highest improvements in weight gain (26%) and feed intake (19%), the latter comparing statistically to the 10% FM PC diet. The HI-PRO diet had the best FCR (1.88), again comparable to the PC (1.73). The NO-PRO diet had the highest protein, P, lipid and energy digestibility, suggesting no additive effect of protease on these coefficients. In terms of gut histomorphology, the LO-PRO and PC diets had the highest measurements and were statistically similar which may have explained similarities in feed intake. Compared to the NC, the HI-PRO diet produced the highest level of change in net profit due to gains in feeding efficiency however, the LO-PRO showed better improvements in terms of growth. Based on these findings, the ternary combination of protease with xylanase and phytase (LOPRO) has potential in limiting FM use for tilapia grow-out feeds, however, the economic efficiencies were still below that of a 10% FM diet. Future considerations for research should target the indigestible dietary components in order to optimise enzyme dosages and maximise the benefits of each enzymes. In conclusion, a comparative life cycle assessment (LCA) was used to evaluate the environmental impacts of low FM diets and commercial feeds associated with tilapia production in Thailand. The study showed that the low FM enzyme supplemented diets had lower impact potentials and were environmental superior to the average (10% FM) commercial standard. LCA modules are recommended for least-cost formulation programmes as an option going forward. Additionally, LCA can be used as a predictive tool to guide farmers, especially small-scale producers, on the potential impacts of feed input choices and feeding practices. This will ensure higher product quality but also demonstrate environmental responsibility on the part of aquafeed and fish producers to final seafood consumers.

Enzymes and subcritical water can be used for the extraction of hemicelluloses from cereal by-products, making the processes eco-friendly. The polysaccharides extracted from cereal by-products can be used as matrices for development of materials for various applications. This includes bio-based materials such as films and hydrogels, which offer alternatives to existing materials produced from petrochemicals. The polymeric structure of cereal hemicelluloses contains functional groups which enable the modification of their structure by cross-linking, resulting in the formation of hydrogels. This project aims to use subcritical water extraction (SWE) to extract arabinoxylans (AXs) from corn and rye bran meanwhile the enzymatic treatment is done for purifying the samples during both pre- and post-treatment. AXs were further crosslinked by enzyme (laccase) for hydrogel preparation. During the whole project, the characterization included moisture and yield determination, starch and protein content which were tested using a spectrophotometer, monosaccharide content was analyzed by high performance anion exchange chromatography followed by pulsed amperometric detection (HPAEC-PAD) and phenolic acid content was quantified by high performance liquid chromatography (HPLC). The pretreatment for destarching and SWE process was successful. The result showed that arabinoxylans form corn bran were having higher content of arabino substituents, arabino toxylans ratio and ferulic acid content than rye samples. The enzymatic crosslinking could form strong gels in the condition that the AXs had high ferulic acid content. In terms of forming strong hydrogels or to improving the properties of AXs gel, the pre- and post-treatment should be optimized to increase the purity of the extracted feruloylated AX content. / Enzymer och subkritiskt vatten kan användas för extraktion av hemicellulosa från spannmålsbiprodukter, vilket gör extraktionen miljövänlig. Polysackariderna extraherade från spannmålsprodukter kan användas som matriser för utveckling av material för diverse applikationer. Detta inkluderar biobaserade material som filmer och hydrogeler, där petrokemikalier kan ersättas som råvara. Den polymera strukturen hos spannmålshemicelluloser innehåller funktionella grupper som möjliggör formation av tvärbindningar vilket resulterar i bildandet av hydrogeler. Syftet med detta projekt är extraktion av arbinoxylaner (AXs) från majs och rågkli genom att använda subkritiskt vatten-extraktion (SWE) där rening under för- och efterbehandling utförs enzymatiskt. AX modifierades därefter enzymatiskt (laccas) med tvärbindningar för hydrogelframställning. Under hela projektet karakteriserades hydrogelen utifrån fuktinnehåll, bestämmelse av utbyte, stärkelse och proteininnehåll som testades med en spektrofotometer, monosackaridhalten analyserades med högpresterande anjonsutbyteskromatografi följt avpuls-amperometrisk detektion (HPAEC-PAD), samt kvantifierades fenolsyrahalten med högupplöst vätskekromatografi (HPLC). Resultatet visade att arabinoxylaner från majskli hade högre innehåll av arabinosubstituenter, där förhållandet mellan arabino och xylans, samt arabino och ferulsyra innehållet var högre än för rågproverna. Den enzymatiska tvärbindningen kunde bilda starka geler i det tillståndet där AX hade en hög ferulsyrahalt. När det gäller att bilda starka hydrogeler eller att förbättra egenskaperna hos AXs-gel, bör för-och efterbehandlingen optimeras för att öka renheten fördet extraherade feruloylerade AX-innehållet.