Interactions of arabinoxylan and (1,3)(1,4)-ß-glucan with cellulose networks.

To identify interactions of relevance to the structure and properties of the primary cell walls of cereals and grasses, we used arabinoxylan and (1,3)(1,4)-ß-glucan, major polymers in cereal/grass primary cell walls, to construct composites with cellulose produced by Gluconacetobacter xylinus. Both polymers associated prolifically with cellulose without becoming rigid or altering the nature or extent of cellulose crystallinity. Mechanical properties were modestly affected compared with xyloglucan or pectin (characteristic components of nongrass primary cell walls) composites with cellulose. In situ depletion of arabinoxylan arabinose side chains within preformed cellulose composites resulted in phase separation, with only limited enhancement of xylan-cellulose interactions. These results suggest that arabinoxylan and (1 → 3)(1 → 4)-ß-d-glucan are not functional homologues for either xyloglucan or pectin in the way they interact with cellulose networks. Association of cell-wall polymers with cellulose driven by entropic amelioration of high energy cellulose/water interfaces should be considered as a third type of interaction within cellulose-based cell walls, in addition to molecular binding (enthalpic driving force) exhibited by, for example, xyloglucans or mannans, and interpenetrating networks based on, for example, pectins.

Biomass attachment and microbiota shifts during porcine faecal in vitro fermentation of almond and macadamia nuts differing in particle sizes.

Nuts are highly nutritious and good sources of dietary fibre, when consumed as part of a healthy human diet. Upon consumption, nut particles of various sizes containing lipids entrapped by the plant cell walls enter the large intestine where they are fermented by the resident microbiota. This study investigated the microbial community shifts during in vitro fermentation of almond and macadamia substrates, of two particle sizes including fine particles (F = 250-500 µm) and cell clusters (CC = 710-1000 µm). The aim was to determine how particle size and biomass attachment altered the microbiota. Over the 48 h fermentation duration, short chain fatty acid concentrations increased due to particle size rather than nut type (almond or macadamia). However, nut type did change microbial population dynamics by stimulating specific genera. Tyzzerella, p253418B5 gut group, Lachnospiraceae UCG001, Geotrichum, Enterococcus, Amnipila and Acetitomaculum genera were unique for almonds. For macadamia, three unique genera including Prevotellaceae UCG004, Candidatus Methanomethylophilus and Alistipes were noted. Distinct shifts in the attached microbial biomass were noted due to nut particle size. Bacterial attachment to nut particles was visualised in situ during fermentation, revealing a decrease in lipids and an increase in attached bacteria over time. This interaction may be a pre-requisite for lipid breakdown during nut particle disappearance. Overall, this study provides insights into how nut fermentation alters the gut microbiota and the possible role that gut microbes have in lipid degradation.

Differential effects of pectin-based dietary fibre type and gut microbiota composition on in vitro fermentation outcomes.

Interactions between human gut microbiota and dietary fibres (DF) are influenced by the complexity and diversity of both individual microbiota and sources of DF. Based on 480 in vitro fermentations, a full factorial experiment was performed with six faecal inocula representing two enterotypes and three DF sources with nanometer, micrometer, and millimeter length-scales (apple pectin, apple cell walls and apple particles) at two concentrations. Increasing DF size reduced substrate disappearance and fermentation rates but not biomass growth. Concentrated DF enhanced butyrate production and lactate cross-feeding. Enterotype differentiated final microbial compositions but not biomass or fermentation metabolite profiles. Individual donor microbiota differences did not influence DF type or concentration effects but were manifested in the promotion of different functional microbes within each population with the capacity to degrade the DF substrates. Overall, consistent effects (independent of donor microbiota variation) of DF type and concentration on kinetics of substrate degradation, microbial biomass production, gas kinetics and metabolite profiles were found, which can form the basis for informed design of DF for desired rates/sites and consequences of gut fermentation. These results add further evidence to the concept that, despite variations between individuals, the human gut microbiota represents a community with conserved emergent properties.

Bringing back a forgotten legume-Sensory profiles of Australian native wattleseeds reveal potential for novel food applications.

Documented as one of the oldest living civilizations, there is now evidence that Indigenous communities in Australia followed a sustainable lifestyle with well-designed agricultural practices and adequate physical activity. Commonly known as wattleseeds in Australia, unique cultivars of Acacia have been consumed by Indigenous Australians for over 60,000 years. This research used descriptive sensory profiling to develop a lexicon for the aroma and flavor profiles of four wattleseed species before and after being subjected to different processing techniques. The processing methods selected were pressure cooking, dry roasting, wet roasting, and malting. The species included were Acacia kempeana, Acacia adsurgens, Acacia colei, and Acacia victoriae. Sensory differences were observed between the different cultivars as well as between the different food processing techniques. Results show that wattleseed species diversity is a key driver in determining the aroma profile, while taste profiles are modified by the type of processing method applied. PRACTICAL APPLICATION: This study provides foundational knowledge on these culturally significant seeds, supporting practical opportunities to diversify the uses of wattleseeds in food products.

Plant cell wall composition modulates the gut microbiota and metabolites in in-vitro fermentation.

This research investigated the effect of different types of plant cell wall fibres, including cereal (i.e., barley, sorghum, and rice), legume (i.e., pea, faba bean, and mung bean), and tuber (potato, sweet potato, and yam) cell wall fibres on in vitro faecal fermentation profiles and gut microbiota composition. The cell wall composition, specifically the content of lignin and pectin, was found to have a significant influence on the gut microbiota and fermentation outcomes. Compared with type I cell walls (legume and tuber) which have high pectin content, the type II cell walls (cereal) which are high in lignin but low in pectin had a lower fermentation rates and less short-chain fatty acid production. The redundancy analysis showed samples with similar fibre composition and fermentation profiles clustered together, and the principal coordinate analysis revealed separation among different types of cell walls and closer proximity among the same cell wall types. These findings emphasize the importance of cell wall composition in shaping the microbial community during fermentation and contribute to a better understanding of the relationship between plant cell walls and gut health. This research has practical implications for the development of functional foods and dietary interventions.

Starch structure and nutritional functionality - Past revelations and future prospects.

Starch exists naturally as insoluble semi-crystalline granules assembled by amylose and amylopectin. Acknowledging the pioneers, we have reviewed the major accomplishments in the area of starch structure from the early 18th century and further established the relation of starch structure to nutritional functionality. Although a huge array of work is reported in the area, the review identified that some features of starch are still not fully understood and needs further elucidation. With the rise of diet-related diseases, it has never been more important to understand starch structure and use that knowledge to improve the nutritional value of the world's principal energy source.

In vitro fermentation profiles of undigested fractions from legume and nut particles are affected by particle cohesion and entrapped macronutrients.

Insoluble undigested food residues are the predominant dietary form of 'fibre' from food plants, with the potential for fermentation by microbial species resident within the large intestine. Here we present results on in vitro fermentation of undigested fractions of legumes (chickpea flour, lentil flour, mung bean flour), and nuts (peanut, almond, macadamia) using a pooled faecal inoculum from pigs fed a nut- and legume-free diet. All substrates were pre-digested in vitro. Nuts were also separated into two particle sizes (PS), cell cluster (CC = 710-1000 µm) and fine (F = 250-500 µm), to test the effect of PS. All substrates tested were fermented for 48 hours, and measured according to gas production, with lentil (within legume flours) being the highest gas producer, and peanut being the highest gas producer within nuts. Undigested fractions from Nuts_F had significantly higher gas production than those from Nuts_CC, consistent with differences in surface area between the two PS. Relative short chain fatty acid concentrations between samples as metabolite end-products were consistent with relative gas production. Analysis of unfermented residues after different fermentation times, showed that cellular integrity was a major factor controlling fermentation rates and that entrapped protein/starch (legumes) and lipid (nuts) all contributed to the fermentation outcomes.

Effect of processing on the solubility and molecular size of oat ß-glucan and consequences for starch digestibility of oat-fortified noodles.

White wheat salted noodles containing oats have a slower digestion rate those without oats, with potential health benefits. Oat ß-glucan may play an important role in this. Effects of sheeting and shearing during noodle-making and subsequent cooking on ß-glucan concentration, solubility, molecular size and starch digestibility were investigated. The levels of ß-glucan were reduced by 16% after cooking, due to the loss of ß-glucan into the cooking water. Both the noodle-making process and cooking increased the solubility of ß-glucan but did not change its average molecular size. Digestion profiles show that ß-glucan in wholemeal oat flour did not change starch digestion rates compared with isolated starch, but reduced the starch digestion rate of oat-fortified wheat noodles compared to the control (wheat noodles). Confocal laser scanning microscopy suggests that interaction between ß-glucan and protein contributes to the starch-protein matrix and changes noodle microstructure, and thus alters their digestibility.

Amorphous packing of amylose and elongated branches linked to the enzymatic resistance of high-amylose wheat starch granules.

To elucidate starch structural features underlying resistant starch formation, wheat starch granules with three (A-, B- and C- type) crystalline polymorphisms and a range of amylose contents were digested in vitro. The changes in multi-level structure of digestion residues were compared. In the residues of A- and C-type starches, the molecular fine structure (distributions of chain length and whole molecular size), as analyzed by size exclusion chromatography (SEC), remained similar during digestion. In contrast, B-type high amylose wheat starch (HAWS) showed distinct changes in multi-level structures of digestion-resistant fractions: (1) the peak of longer amylopectin branches shifted to a lower degree of polymerization (40 DP); (2) production of α-limit dextrin (~2 nm hydrodynamic radius) in the residues; (3) a small increase of double helix content during digestion, in contrast to 6 % reduction for the A-type starch; (4) a decrease (6 °C lower) in the melting temperature of amylose-lipid complexes. The comparison suggests that elongated branches in B-type starch contribute to the formation of resistant fraction (including α-limit dextrin) against α-amylase. The amorphous packing of starch polymers with elongated branches together with the absence of surface pores and channels is proposed to be the basis for the enzymatic resistance of granular HAWS.

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing.

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Wheat cell walls and constituent polysaccharides induce similar microbiota profiles upon in vitro fermentation despite different short chain fatty acid end-product levels.

Plant cell walls as well as their component polysaccharides in foods can be utilized to alter and maintain a beneficial human gut microbiota, but it is not known whether the architecture of the cell wall influences the gut microbiota population. In this study, wheat flour cell walls (WCW) were isolated and compared with their major constituents - arabinoxylan (AX), mixed linkage (1,3)(1,4)-ß-glucan (MLG) and cellulose - both separately and as a physical mixture of polysaccharides (Mix) equivalent in composition to WCW. These samples underwent in vitro fermentation with a faecal inoculum from pigs fed a diet free of cereals and soluble-fibre to avoid prior adaptation to substrates. During fermentation, samples were collected for DNA extraction and 16S rRNA gene amplicon sequencing. Bioinformatics analyses revealed that the microbial communities promoted during fermentation by AX, MLG, Mix and WCW were similar at the genus level, but differed from the microbiota observed for the cellulose substrate. Differences in proportions of propionate and butyrate end-products were associated with differences in the relative levels of genera. These findings show that, in this experiment, the microbes that flourished were able to utilize diverse WCW polysaccharides alone, in mixtures or in intact cell walls in a similar way, but that different fermentation end-products were associated with AX (propionate) or MLG (butyrate) polysaccharides.

Interaction of cellulose and xyloglucan influences in vitro fermentation outcomes.

To investigate the effects of interactions between cellulose and xyloglucan (XG) on in vitro fermentation, a composite of bacterial cellulose (BC) incorporating XG during pellicle formation (BCXG), was fermented using a human faecal inoculum, and compared with BC, XG and a mixture (BC&XG) physically blended to have the same BC to XG ratio of BCXG. Compared to individual polysaccharides, the fermentation extent of BC and fermentation rate of XG were promoted in BC&XG. XG embedded in the BCXG composite was degraded less than in BC&XG, while more cellulose in BCXG was fermented than in BC&XG. This combination explains the similar amount of short chain fatty acid production noted throughout the fermentation process for BCXG and BC&XG. Microbial community dynamics for each substrate were consistent with the corresponding polysaccharide degradation. Thus, interactions between cellulose and XG are shown to influence their fermentability in multiple ways.

In vitro fermentation outcomes of arabinoxylan and galactoxyloglucan depend on fecal inoculum more than substrate chemistry.

Using in vitro fermentation conditions, this study investigated the fermentation characteristics of arabinoxylan (AX) and xyloglucan (XG) with a fecal inoculum that was collected either from humans consuming unrestricted diets or pigs fed a semi-defined diet with cellulose being the sole non-starch polysaccharide for 10 days prior to fecal collection. Metagenomic analysis revealed that microbial communities in the two types of inoculum were distinctively different, which led to distinct fermentation characteristics with the polysaccharides. The microbial communities fermented with the porcine fecal inoculum were clustered according to the fermentation time, while those fermented with the human fecal inoculum were differentiated by the substrates. Using the porcine fecal inoculum, irrespective of the substrates, Prevotella copri and the unclassified lineage rc4-4 were the dominant operational taxonomic units (OTUs) promoted during fermentation. Fermentation of wheat AX (WAX) and galacto-XG (GXG) with the human fecal inoculum, however, promoted different OTUs, except for a shared OTU belonging to Lachnospiraceae. Specifically, WAX promoted the growth of Bacteroides plebeius and a Blautia sp., while GXG promoted an unclassified Bacteroidales, Parabacteroides distasonis, Bacteroides uniformis and Bacteroides sp. 2. These changes in bacterial communities were in accordance with the short chain fatty acid (SCFA) production, where comparable SCFA profiles were obtained from the porcine fecal fermentation while different amounts and proportions of SCFA were acquired from fermentation of WAX and GXG with the human fecal inoculum. Altogether, this study indicated that the starting inoculum composition had a greater effect than polysaccharide chemistry in driving fermentation outcomes.

Metabolism of Black Carrot Polyphenols during In Vitro Fermentation is Not Affected by Cellulose or Cell Wall Association.

Fruit and vegetable polyphenols are associated with health benefits, and those not absorbed could be fermented by the gastro-intestinal tract microbiota. Many fermentation studies focus on "pure" polyphenols, rather than those associated with plant cell walls (PCW). Black carrots (BlkC), are an ideal model plant food as their polyphenols bind to PCW with minimal release after gastro-intestinal digestion. BlkC were fractionated into three components-supernatant, pellet after centrifugation, and whole puree. Bacterial cellulose (BCell) was soaked in supernatant (BCell&S) as a model substrate. All substrates were fermented in vitro with a pig faecal inoculum. Gas kinetics, short chain fatty acids, and ammonium production, and changes in anthocyanins and phenolic acids were compared. This study showed that metabolism of BlkC polyphenols during in vitro fermentation was not affected by cellulose/cell wall association. In addition, BCell&S is an appropriate model to represent BlkC fermentation, suggesting the potential to examine fermentability of PCW-associated polyphenols in other fruits/vegetables.

Wood hemicelluloses exert distinct biomechanical contributions to cellulose fibrillar networks.

Hemicelluloses, a family of heterogeneous polysaccharides with complex molecular structures, constitute a fundamental component of lignocellulosic biomass. However, the contribution of each hemicellulose type to the mechanical properties of secondary plant cell walls remains elusive. Here we homogeneously incorporate different combinations of extracted and purified hemicelluloses (xylans and glucomannans) from softwood and hardwood species into self-assembled networks during cellulose biosynthesis in a bacterial model, without altering the morphology and the crystallinity of the cellulose bundles. These composite hydrogels can be therefore envisioned as models of secondary plant cell walls prior to lignification. The incorporated hemicelluloses exhibit both a rigid phase having close interactions with cellulose, together with a flexible phase contributing to the multiscale architecture of the bacterial cellulose hydrogels. The wood hemicelluloses exhibit distinct biomechanical contributions, with glucomannans increasing the elastic modulus in compression, and xylans contributing to a dramatic increase of the elongation at break under tension. These diverging effects cannot be explained solely from the nature of their direct interactions with cellulose, but can be related to the distinct molecular structure of wood xylans and mannans, the multiphase architecture of the hydrogels and the aggregative effects amongst hemicellulose-coated fibrils. Our study contributes to understanding the specific roles of wood xylans and glucomannans in the biomechanical integrity of secondary cell walls in tension and compression and has significance for the development of lignocellulosic materials with controlled assembly and tailored mechanical properties.

"Dietary fibre": moving beyond the "soluble/insoluble" classification for monogastric nutrition, with an emphasis on humans and pigs.

This review describes dietary fibres originating from a range of foods, particularly in relation to their plant cell walls. It explores the categorization of dietary fibres into "soluble" or "insoluble". It also emphasizes dietary fibre fermentability, in terms of describing how the gastro-intestinal tract (GIT) microbiota respond to a selection of fibres from these categories. Food is categorized into cereals, legumes, fruits and vegetables. Mention is also made of example whole foods and why differences in physico-chemical characteristics between "purified" and "non-purified" food components are important in terms of health. Lastly, recommendations are made as to how dietary fibre could be classified differently, in relation to its functionality in terms of fermentability, rather than only its solubility.

Molecular rearrangement of starch during in vitro digestion: toward a better understanding of enzyme resistant starch formation in processed starches.

Resistant starch (RS) is defined as the fraction of starch that escapes digestion in the small intestine, serving as a fermentation substrate for beneficial colonic bacteria. Several studies have been focused on the description of the RS fractions from different starch varieties, but little attention has been paid to the digestion process itself that, from the present work, seems to play a key role in the generation of enzyme-RS (ERS), as determined in vitro. High-amylose starch samples, extruded at two different processing conditions, have been characterized at different stages of in vitro digestion using scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), infrared spectroscopy (FT-IR), solid state (13)C NMR spectroscopy, and X-ray diffraction (XRD). Control samples kept for 18 h in the digestion solution without starch hydrolyzing enzymes (alpha-amylase and amyloglucosidase) were used for comparison purposes. An increase in molecular order was favored by the hydrolytic action of the enzymes, reflected in an increase in double helical order observed by NMR, higher crystallinity measured by XRD, and corresponding changes in FT-IR spectra. An increase in the intensity of the scattering objects was also observed by SAXS as a function of digestion. SAXS from the dry ERS fractions reveals the 001 reflection of crystallites formed during the digestion process, corresponding to a characteristic dimension of the resistant crystalline fraction of approximately 5 nm. The changes found suggest that enzyme resistant starch does not refer to a specific structure present in predigested starches, but may in fact be formed during the digestion process through the rearrangement of amylose chains into enzyme-resistant structures of higher crystallinity. Therefore, the resistance to enzyme digestion of a specific processed starch is the result of a competition between the kinetics of enzyme hydrolysis and the kinetics of amylose retrogradation.

Mechanisms of utilisation of arabinoxylans by a porcine faecal inoculum: competition and co-operation.

Recent studies show that a single or small number of intestinal microbes can completely degrade complex carbohydrates. This suggests a drive towards competitive utilisation of dietary complex carbohydrates resulting in limited microbial diversity, at odds with the health benefits associated with a diverse microbiome. This study investigates the enzymatic metabolism of wheat and rye arabinoxylans (AX) using in vitro fermentation, with a porcine faecal inoculum. Through studying the activity of AX-degrading enzymes and the structural changes of residual AX during fermentation, we show that the AX-degrading enzymes are mainly cell-associated, which enables the microbes to utilise the AX competitively. However, potential for cross-feeding is also demonstrated to occur by two distinct mechanisms: (1) release of AX after partial degradation by cell-associated enzymes, and (2) release of enzymes during biomass turnover, indicative of co-operative AX degradation. This study provides a model for the combined competitive-co-operative utilisation of complex dietary carbohydrates by gut microorganisms.

Extracellular depolymerisation triggers fermentation of tamarind xyloglucan and wheat arabinoxylan by a porcine faecal inoculum.

Arabinoxylan (AX) and xyloglucan (XG) are important components of primary cell walls of cereal grains and vegetables/fruits, respectively. Despite the established health benefits of these non-starch polysaccharides, the mechanisms of their utilisation by the gut microbiota are poorly understood. In this study, the mechanisms of solubilised wheat AX and tamarind XG degradation were investigated under in vitro fermentation conditions using a porcine faecal inoculum. Through structural analysis of the polymers, we demonstrate that depolymerisation by microbial surface accessible endo-degrading enzymes occurs prior to active fermentation of AX or XG. Breakdown products are released into the medium and potentially utilised cooperatively by other microbes. Acetate and propionate are the main fermentation products and are produced concurrently with polysaccharide depletion. Butyrate, however, is produced more slowly consistent with it being a secondary metabolite.

Quantitative structural organisation model for wheat endosperm cell walls: Cellulose as an important constituent.

The cell walls of cereal endosperms are a major source of fibre in many diets and of importance in seed structure and germination. Cell walls were isolated from both pure wheat endosperm and milled flour. 13C CP/MAS NMR in conjunction with methylation analysis before and after acid hydrolysis showed that, in addition to arabinoxylan (AX) and (1, 3; 1, 4)-ß-D-glucan (MLG), wheat endosperm cell walls contain a significant proportion of cellulose (ca 20%) which is tightly bound to xylans and mannans. Light microscopy showed that the cellulose was relatively evenly distributed across the grain endosperm. The cell walls contain a fibrous acid-resistant core structure laminated by matrix polysaccharides as revealed by AFM imaging. A model for endosperm cell wall structural organisation is proposed, based on a core of cellulose and interacting non-cellulosic polysaccharides which anchors AX (with very occasional diferulic acid cross-linking) that in turn retains MLGs through physical entanglement.