Master of Science / Food Science Institute / Fadi Aramouni / β-(1,4)-endoxylanases, commonly referred to as xylanases, have become integral to the industrial breadmaking process. This enzyme is known to cause improvement in dough rheology, loaf volume, and crumb grain. Significant research has been conducted regarding the structure, function, and inhibition of xylanases, but there is currently no quick and reproducible method to evaluate their effect in baking. The goal of this research was to develop a quantitative method for this purpose and to determine why the effect of xylanases varies with different wheat flours. The currently used methods of test baking, dough stickiness, and spectrophotometric analysis for reducing sugars were evaluated, and failed to provide reproducible results. Therefore, a new method was developed to measure the Flour Water Expression Rate (FWER) with the addition of xylanases. Commercially available enzymes from Aspergillus niger and Bacillus subtilis were evaluated in this study. The FWER method measures the amount of water released by the xylanase over a set period of time. This method consistently provided statistically significant data (p<0.05), which was able to provide a comparison of xylanases from A. niger and B. subtilis in different flours. The results indicated that the xylanase from A. niger tends to release more water, have a higher FWER value, than the xylanase from B. subtilis. In one flour, A. niger xylanase resulted in an FWER of 15.18 compared to B. subtilis xylanase that resulted in an FWER of 9.57 at equivalent activities. However, inhibitors in the wheat appeared to cause an impact on the FWER, which was evaluated with an uninhibited xylanase from B. subtilis. This new method for the evaluation of xylanases in baking suggests varying levels of xylanase inhibitors in wheat may be the reason xylanases effect wheat flours differently.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18318号 / 農博第2043号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4825(農学部図書室) / 31176 / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 星野 敏, 教授 縄田 栄治 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Cellulose and hemicelluloses have attracted increasing interest as renewable biopolymers because of their abundance. Furthermore, the recognition of biomass as a sustainable and renewable source of biofuels has driven research into the assembly and disassembly of polymers within plant cell walls. Cellulose thin films are useful in the study of interactions between cellulose and hemicelluloses, and quartz crystal microbalances with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM) are widely used to investigate polymer adsorption/desorption at liquid/solid interfaces. In this study, smooth trimethylsilyl cellulose (TMSC) films were spincoated onto gold QCM-D sensors and hydrolyzed into ultrathin cellulose films upon exposure to aqueous HCl vapor. The adsorption of arabinoglucuronoxylan (AGX) and arabinoxylan (AX) onto these cellulose surfaces was studied. The effects of structure, molar mass and ionic strength of the solution were considered. Increasing ionic strength increased AGX and AX adsorption onto cellulose. While AGX showed greater adsorption onto cellulose than AX by QCM-D, the trend was reversed in SPR experiments. The combination of QCM-D and SPR data showed a greater amount of water was trapped within the AX films. Both adsorbed AGX and AX films were subsequently visualized by AFM. Images from AFM showed AGX and AX adsorbed as aggregates from water, while AGX and AX adsorbed from CaCl2 yielded smaller xylan particles with more numerous globular structures on the cellulose surfaces. Images from AFM of xylan films on bare gold surfaces also showed layers of uniform aggregates that were consistent with AX and AGX aggregation in solution. / Master of Science

Cette étude a pour objectif d'évaluer la relation entre la structure (au niveau physique et / ou moléculaire) de son de blé et de la couche à aleurone et leurs effets santé. En gardant la composition du son et de l'aleurone constante, leur structures ont été modifiés par des traitements mécaniques et / ou enzymatiques de façon à casser leur structure matricielle complexe et d'augmenter la bioaccessibilité des leurs fibres alimentaires et composés phénoliques. La capacité antioxydante des ces fractions a été évaluée par une méthode standard in vitro et par un modèle de digestion gastrique in vitro. La déstructuration physique du son et aleurone par le broyage a augmenté leur surface spécifique conduisant à une plus grande exposition du groupement acide férulique (AF) qui a augmenté leur capacité antioxydante. Le traitement enzymatique réalisée sur aleurone a modifié son organisation moléculaire, qui a largement libéré l'AF des arabinoxylanes (AX). Cette destructuration a apporté une amélioration plus importante sur la capacité antioxydante de l'aleurone que celle mené par le broyage. Comme l'aleurone est riche en AX fermentescibles, les effets de sa structure plus accessible et avec une plus grande dépolymérisation ont été évaluées en utilisant un modèle in vitro en mimant le côlon humain. La déstructuration physique de l'aleurone par le broyage n'a pas amélioré leur pouvoir fermentescible, c'est à dire que le métabolisme de composés phénoliques et de la formation des chaînes courtes d'acides gras ont été comparable à ceux obtenus pour l'aleurone natif. Néanmoins, la dégradation enzymatique de l'aleurone en apportant une plus grande quantité des AX soluble et de l'AF biodisponible a augmenté le métabolisme de l'AF dans le colon dans des métabolites avec propriétés anti-inflammatoires. Une étude in vivo avec des souris sur un régime riche en graisses a été fait pour évaluer le potentiel de l'aleurone physiquement et moléculaire déstructuré pour neutraliser les désordres métaboliques tels que l'obésité, le stress oxydatif et l'inflammation. La déstructuration physique de l'aleurone n'a pas eu d'effet positif sur les désordres métaboliques. La fraction d'aleurone qui a présenté la plus grande quantité d'AX soluble et de l'AF biodisponible a bien réduit l'obésité (gain de poids, l'adiposité et la sécrétion de leptine) et la résistance à l'insuline chez la souris. Par contre, aucune différence significative n'a été observée dans le stress oxydatif et l'inflammation des souris nourries avec des régimes enrichis en aleurone. En conclusion, nous avons démontré que les effets santé de l'aleurone sont clairement liées à l'intégrité de sa structure. Ses effets santé ont été accrues par une modification de sa structure principalement au niveau moléculaire en dégradant ses parois cellulaire pour augmenter la bioaccessibilité et la biodisponibilité des composés nutritionnellement intéressants, tels que l'AX et l'FA. / The objective of this study was to evaluate the relationship between the structure (at physical and/or molecular level) of wheat bran and aleurone and their health effects. Keeping the composition of bran and aleurone constant, their structure was modified by mechanical and/or enzymatic treatments in order to disrupt their complex matrix structure and increase the bioaccessibility of their dietary fibre and phenolic compounds. The antioxidant capacity of the bran and aleurone fractions was evaluated by a standard in vitro test and by an in vitro gastric digestion model. The grinding disrupted the physical structure of bran and aleurone increasing their specific surface leading to a greater exposition of ferulic acid moiety (FA) which increased their antioxidant capacity. Enzymatic treatment performed on aleurone acted on its molecular organisation releasing its FA from arabinoxylans (AX). This destructuration improved the antioxidant capacity of aleurone even more than the one allowed by grinding. As aleurone is rich in highly fermentable AX, the effects of its better accessibility and depolymerisation were evaluated using an in vitro model mimicking the human colon. The physical destructuration of aleurone by grinding did not improve its fermentability, i.e. the colonic metabolism of phenolic compounds and the formation of short-chain fatty acids were similar compared to the native aleurone. Nevertheless, the enzymatic degradation of aleurone produced higher amount of soluble AX and bioavailable FA improving the metabolism of colonic FA in metabolites with anti-inflammatory properties. An in vivo mouse study with a high-fat diet was used to evaluate the potential of physically and molecularly destructured aleurone to counteract the metabolic disorders, such as obesity, oxidative stress and inflammation. The physical destructuration of aleurone did not have any positive effect on the metabolic disorders. The aleurone fraction, which presented the highest level of soluble AX and bioavailable FA, reduced the obesity (body weight gain, adiposity, and leptin secretion) and insulin resistance in mice. But no significant differences were observed in the oxidative stress and inflammation status of mice fed with any of the aleurone-based diets. In conclusion, we demonstrated that the aleurone health effects were clearly linked to the integrity of its structure. Its health effects have been increased by modification of its structure mainly at molecular level by degrading its cell wall to increase the bioaccessibility and bioavailability of nutritionally interesting compounds, such as AX and FA.

Arabinoxylan ferulate representing a macromolecular LCC model valid for annual plants is synthesized under Mitsunobu conditions. The content of ferulic acid ester is tuned by the reaction conditions achieving degree of substitution values from 0.09 to 0.45. Utilization of the chloride-free solvent N-methyl-2-pyrrolidone allows the design of pure Mitsunobu products without occurrence of deoxychloro moieties arising from Appel type reaction. 2D NMR experiments reveal nature-identical structure of ferulate moieties present at position 5 of the arabinose side chain. Enzymatic dehydrogenation polymerization of coniferyl alcohol on ferulate anchor groups under homogeneous conditions lead to β-O-4, β-5, and Hibbert ketone structures identified by Py-GC-MS. The results are valuable to study structure-property relationships within the formation of natural and non-native lignins.

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.

Biopolymers are becoming more attractive as advanced wound dressings because of their naturally derived origin, abundance, low cost and high compatibility with the wound environment. Arabinoxylan (AX) is a class of polysaccharide polymers derived from cereal grains that are primarily used in food products and cosmetic additives. Its application as a wound dressing material has yet to be realized. In this two-pronged project, arabinoxylan ferulate (AXF) was fabricated into electrospun fibers and gel foams to be evaluated as platforms for wound dressing materials. In the first study, AXF was electrospun with varying amounts of gelatin. In the second study, AXF was dissolved in water, enzymatically crosslinked and lyophilized to form gel foams. The morphology, mechanical properties, porosity, drug release kinetics, fibroblast cell response and anti-microbial properties were examined for both platforms. Carbohydrate assay was conducted to validate the presence of arabinoxylan ferulate in the electrospun GEL-AXF fibers. Swelling and endotoxin quantification studies were done to evaluate the absorptive capacity and sterilization agent efficacy respectively in AXF foams. The results indicated successful fabrication of both platforms which validated the porous, absorptive, biocompatibility and drug release properties. The results also exhibited that silver impregnated AXF scaffolds inhibited growth of Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis bacteria species, anti-microbial properties necessary to function as advanced wound dressing materials. Future work will be done to improve the stability of both platforms as well as evaluate its applications in vivo.

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed

A parede celular das plantas inclui componentes polissacarídeos complexos, e a sacarificação destes polímeros necessita da ação de conjuntos de enzimas que atuem em sinergia. Enzimas podem formar complexos multi-enzimáticos que possuem mais de uma atividade catalítica derivada de domínios distintos de uma mesma cadeia polipeptídica. O objetivo deste trabalho foi construir uma enzima bifuncional com os domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) para desconstrução de material lignocelulósico de cana-de-açúcar. Para isso, dois diferentes domínios catalíticos: acetilxilano esterase (Axe) e feruloil esterase (Fae) oriundos de Clostridium thermocellum foram fundidas para criar a quimera feruloil esterase/acetil-xilano esterase (FaeAxe). O desenho racional da quimera foi feito utilizando-se de métodos computacionais, que permitiram a criação de um modelo estrutural da enzima. A construção da quimera foi feita por overlap PCR, clonada em vetor pET-SUMO e expressa em Escherichia coli. As duas enzimas parentais (Fae e Axe) foram clonadas em vetor pET28 e expressas em E. coli. Durante a etapa de expressão, observou-se que todas as enzimas foram expressas na forma solúvel. As enzimas feruloil esterase e acetilxilano esterase têm como substrato o polímero arabinoxilano, cuja degradação é uma etapa chave na sacarificação de biomassa. Dessa forma, as atividades da quimera, bem como das enzimas parentais foram testadas contra polímeros arabinoxilano de trigo e arabinoxilano de cana-de-açúcar após a hidrólise pela endoxilanase GH11 de Bacillus Subtilis e analisadas por meio de espectrometria de massas. A atividade desacetilase da enzima parental acetil-xilano esterase e da quimera FaeAxe foram confirmadas, evidenciando que a quimera preservou essa atividade catalítica. A atividade da enzima feruloil esterase e da quimera FaeAxe na remoção de ácido ferúlico dos oligossacarídeos gerados pela endoxilanase GH11 não foi observada / The plant cell wall is comprised of a matrix of polysaccharides and saccharification of these polymers requires the joint action of diverse enzymes. Enzymes may form multi-enzymatic complexes that have more than one catalytic activity derived from different domains of a single polypeptide chain. The aim of this work was to construct a bifunctional enzyme with two catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) for degradation of sugar cane lignocellulosic material. The two different catalytic domains: acetylxylan esterase (Axe) and feruloyl esterase (Fae) from Clostridium thermocellum were fused to generate a bifunctional chimera feruloyl esterase/acetylxylan esterase (FaeAxe). A molecular model was created by rational design using a 3D-structure guided strategy. The fusion was created using overlap PCR, and the resulting product was cloned into the pETSUMO vector. The chimeric protein and the parental enzymes were expressed in Escherichia coli and purified and the enzymes were expressed in soluble form. Xylanases, feruloyl esterases and acetylxylan esterases degrade arabinoxylan polymers and their activity is a key step in the saccharification of biomass. The catalytic properties of the chimera and of the parental enzymes were tested against wheat and sugarcane arabinoxylan polymers after hydrolysis by GH11 endoxylanase from Bacillus subtilis and analyzed by mass spectroscopy. The deacetylase activity of acetyl-xylan esterase parental enzyme and FaeAxe chimera were confirmed, showing that the chimera kept the deacetylase activity. After hydrolysis by GH11 endoxylanase from Bacillus subtilis the feruloyl esterase and FaeAxe chimera activities on ferulic acid removal were not observed