Independent fermentation and metabolism of dietary polyphenols associated with a plant cell wall model.

Ingested polyphenols from plant-based foods are in part carried to the large intestine and metabolised by resident microbiota. This work investigated the release and microbial transformation of polyphenols adsorbed individually or in combination to apple cell walls (ACW) and pure (bacterial) cellulose (BC). BC and ACW, representing poorly- and highly-fermentable fibre models respectively, were used to investigate influences of interactions with polyphenols (cyanidin-3-glucoside, (±)-catechin, ferulic acid), on the release and microbial metabolism of polyphenols during in vitro digestion and fermentation. Bound polyphenols were partially released (20-70%) during simulated digestion, depending on polyphenol molecular structure. All remaining bound polyphenols were completely released and metabolised after 6-9 h by porcine large intestine microbiota, with formation of a number of intermediates and end-products. The same pathways of polyphenol microbial metabolism were observed in the presence and absence of ACW/BC, suggesting that microbial metabolism of polyphenols and carbohydrate substrates seems likely independent. Some polyphenol metabolism products were produced faster in the presence of carbohydrate fermentation, particularly of ACW. Microbial metabolism pathways of model polyphenols by a porcine faecal inoculum are not affected by being associated with BC or ACW, but the rate of metabolism is modestly enhanced with concurrent carbohydrate fermentation.

Review: Effects of fibre, grain starch digestion rate and the ileal brake on voluntary feed intake in pigs.

Grains rich in starch constitute the primary source of energy for both pigs and humans, but there is incomplete understanding of physiological mechanisms that determine the extent of digestion of grain starch in monogastric animals including pigs and humans. Slow digestion of starch to produce glucose in the small intestine (SI) leads to undigested starch escaping to the large intestine where it is fermented to produce short-chain fatty acids. Glucose generated from starch provides more energy than short-chain fatty acids for normal metabolism and growth in monogastrics. While incomplete digestion of starch leads to underutilised feed in pigs and economic losses, it is desirable in human nutrition to maintain consistent body weight in adults. Undigested nutrients reaching the ileum may trigger the ileal brake, and fermentation of undigested nutrients or fibre in the large intestine triggers the colonic brake. These intestinal brakes reduce the passage rate in an attempt to maximise nutrient utilisation, and lead to increased satiety that may reduce feed intake. The three physiological mechanisms that control grain digestion and feed intake are: (1) gastric emptying rate; (2) interplay of grain digestion and passage rate in the SI controlling the activation of the ileal brake; and (3) fermentation of undigested nutrients or fibre in the large intestine activating the colonic brake. Fibre plays an important role in influencing these mechanisms and the extent of their effects. In this review, an account of the physiological mechanisms controlling the passage rate, feed intake and enzymatic digestion of grains is presented: (1) to evaluate the merits of recently developed methods of grain/starch digestion for application purposes; and (2) to identify opportunities for future research to advance our understanding of how the combination of controlled grain digestion and fibre content can be manipulated to physiologically influence satiety and food intake.

Complexity and health functionality of plant cell wall fibers from fruits and vegetables.

The prevalence of lifestyle-related diseases is increasing in developing countries with the causes for death starting to follow the same pattern in the developed world. Lifestyle factors including inadequate dietary intake of fruits and vegetables and over consumption of nutrient-poor processed foods, are considered to be major causal risk factors associated with increased susceptibility to developing certain diseases (Alldrick, 1998 ; Kiani, 2007 ). Recent epidemiological evidence confirms a strong association between dietary fiber and reduced all-cause mortality risk, as well as a risk reduction for a number of non-communicable diseases (Chuang et al., 2012 ). The relationship between dietary fiber and mortality has been described as "convincing observations that call for mechanistic investigations" (Landberg, 2012 ). In particular, the health protective roles played by dietary fibers of different origin are not well understood. Whilst Hippocrates was the earliest known physician to study the health benefits of fiber derived from grains (Burkitt, 1987 ), the functionality of fruit and vegetable fiber, especially in association with other compounds such as polyphenols and carotenoids, is an area of more recent interest. Hence the objective of this review is to assess the complexity and health-related functional role of plant cell wall (PCW) fibers from fruits and vegetables with a particular emphasis on interactions between cell walls and phytonutrients.

Micromechanical model of biphasic biomaterials with internal adhesion: Application to nanocellulose hydrogel composites.

The mechanical properties of hydrated biomaterials are non-recoverable upon unconfined compression if adhesion occurs between the structural components in the material upon fluid loss and apparent plastic behaviour. We explore these micromechanical phenomena by introducing an aggregation force and a critical yield pressure into the constitutive biphasic formulation for transversely isotropic tissues. The underlying hypothesis is that continual fluid pressure build-up during compression temporarily supresses aggregation. Once compression stops and the pressure falls below some critical value, internal aggregation occurs over a time scale comparable to the poroelastic time. We demonstrate this model by predicting the mechanical response of bacterial nanocellulose hydrogel composites, which are promising biomaterials and a structural mimetic for the plant cell wall. Cross-linking of cellulose by xyloglucan creates an extensional resistance and substantially increases the compressive modulus under large compression and densification. In comparison, incorporating non-crosslinking arabinoxylan into the hydrogel has little effect on its mechanics at the strain rates investigated. These results assist in elucidating the mechanical role of these polysaccharides in the complex plant cell wall structure. They also suggest xyloglucan is a suitable candidate to tailor the stiffness of nanocellulose hydrogels in biomaterial design, which includes modulating cell-adhesion in tissue engineering applications. The model and overall approach may be utilised to characterise and design a myriad of biomaterials and mammalian tissues, particularly those with a fibrillar structure. STATEMENT OF SIGNIFICANCE: The mechanical properties of hydrated biomaterials can be non-recoverable upon compression due to increased adhesion occurring between the structural components in the material. Cellulose-hemicellulose composite hydrogels constitute a classical example of this phenomenon, since fibres can freely re-orient and adhere upon fluid loss to produce significant variations in the mechanical response to compression. Here, we model their micromechanics by introducing an aggregation force and a critical yield pressure into the constitutive formulation for transversely isotropic biphasic materials. The resulting model is easy to implement for routine characterization of this type of hydrated biomaterials through unconfined compression testing and produces physically meaningful and reproducible mechanical parameters.

Interpreting atomic force microscopy nanoindentation of hierarchical biological materials using multi-regime analysis.

We present a novel Multi-Regime Analysis (MRA) routine for interpreting force indentation measurements of soft materials using atomic force microscopy. The MRA approach combines both well established and semi-empirical theories of contact mechanics within a single framework to deconvolute highly complex and non-linear force-indentation curves. The fundamental assumption in the present form of the model is that each structural contribution to the mechanical response acts in series with other 'mechanical resistors'. This simplification enables interpretation of the micromechanical properties of materials with hierarchical structures and it allows automated processing of large data sets, which is particularly indispensable for biological systems. We validate the algorithm by demonstrating for the first time that the elastic modulus of polydimethylsiloxane (PDMS) films is accurately predicted from both approach and retraction branches of force-indentation curves. For biological systems with complex hierarchical structures, we show the unique capability of MRA to map the micromechanics of live plant cells, revealing an intricate sequence of mechanical deformations resolved with precision that is unattainable using conventional methods of analysis. We recommend the routine use of MRA to interpret AFM force-indentation measurements for other complex soft materials including mammalian cells, bacteria and nanomaterials.

Micromechanics and poroelasticity of hydrated cellulose networks.

The micromechanics of cellulose hydrogels have been investigated using a new rheological experimental approach, combined with simulation using a poroelastic constitutive model. A series of mechanical compression steps at different strain rates were performed as a function of cellulose hydrogel thickness, combined with small amplitude oscillatory shear after each step to monitor the viscoelasticity of the sample. During compression, bacterial cellulose hydrogels behaved as anisotropic materials with near zero Poisson's ratio. The micromechanics of the hydrogels altered with each compression as water was squeezed out of the structure, and microstructural changes were strain rate-dependent, with increased densification of the cellulose network and increased cellulose fiber aggregation observed for slower compressive strain rates. A transversely isotropic poroelastic model was used to explain the observed micromechanical behavior, showing that the mechanical properties of cellulose networks in aqueous environments are mainly controlled by the rate of water movement within the structure.

Binding of polyphenols to plant cell wall analogues - Part 2: Phenolic acids.

Bacterial cellulose and cellulose-pectin composites were used as well-defined model plant cell wall (PCW) systems to study the interaction between phenolic acids (PA) derived from purple carrot juice concentrate (PCJC) and PCW components. Significant PA depletion from solution occurred, with pure cellulose initially (30s-1h) absorbing more than cellulose-pectin composites in the first hour (ca 20% cf 10-15%), but with all composites absorbing similar levels (ca 30%) after several days. Individual PAs bound to different relative extents with caffeic acid>chlorogenic acid>ferulic acid. Extrapolation of data for these model systems to carrot puree suggests that nutritionally-significant amounts of PAs could bind to cell walls, potentially restricting bioavailability in the small intestine and, as a consequence, delivering PAs to the large intestine for fermentation and metabolism by gut bacteria.

Investigation of the microstructure of milk protein concentrate powders during rehydration: alterations during storage.

The aim of this work was to use scanning electron microscopy to investigate the microstructure of rehydrated milk protein concentrate powder (MPC) particles. A sample preparation method for scanning electron microscopy analysis of rehydrated MPC particles is described and used to characterize the time course of dissolution and the effects of prior storage on the dissolution process. The results show that a combination of different types of interactions (e.g., bridges, direct contact) between casein micelles results in a porous, gel-like structure that restrains the dispersion of individual micelles into the surrounding liquid phase without preventing water penetration and solubilization of nonmicellar components. During storage of the powder, increased interactions occur between and within micelles, leading to compaction of micelles and the formation of a monolayer skin of casein micelles packed close together, the combination of which are proposed to be responsible for the slow dissolution of stored MPC powders.

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524.

AIMS: To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced. METHODS AND RESULTS: Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga. xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3.83 and 3.75 g l(-1) respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2.5 g l(-1). Solid state (13)C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles. CONCLUSIONS: The production rate of bacterial cellulose by Ga. xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features. SIGNIFICANCE AND IMPACT OF THE STUDY: Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga. xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.

Hydrocolloid gel particles: formation, characterization, and application.

Hydrocolloid gel particles of micron and sub-micron size are particularly attractive for use in many applications in the food, agricultural, pharmaceutical, and chemical industries, due to their biocompatibility, perception as "natural" materials, and soft-solid texture. Industrial applications for such particles include uses as texturizers in confectionery and cosmetic products, slow-release encapsulation agents for flavors, nutrients, and pharmaceutical products, and thickeners in soups and sauces. Properties such as particle size, hardness, shape, texture, and molecular release rates can be important for individual applications. In addition, product formats will determine specific needs for physical form (e.g. dry or wet) and compatibility with other components. The diverse range of potential applications for hydrocolloid gel particles provide a driver for understanding-led tailoring of raw material and process conditions. This review introduces some of the materials that are used to form hydrocolloid gel particles and the corresponding gel formation mechanisms. One issue of importance in the production of hydrocolloid gel particles is the control of particle properties, such as release profiles, strength, and detectability within products. An alternative technique to traditional methods of hydrocolloid gel particle production is evaluated and a model for control of particle size, and subsequently other particle properties, is proposed. Key properties of hydrocolloid gel particles are identified and characterization methods for evaluating these properties are described.

Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats.

A bacterially produced cellulose film containing nisin was developed and used in a proof-of-concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of vacuum-packaged frankfurters. Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus K3 in Corn Steep Liquor-Mannitol Medium and were subsequently purified before nisin was incorporated into them. Investigations into the effect of nisin concentrations and contact times on incorporation of nisin into cellulose films showed that the lowest nisin concentration and shortest time needed for production of an effective antimicrobial cellulose film were 625IUml(-1) and 6h, respectively. The active cellulose films produced under these conditions did not, however, significantly reduce L. monocytogenes populations on frankfurters (P>0.05) during refrigerated storage for 14 days as compared to the controls. Films produced using a higher concentration of nisin (2500IUml(-1)) with the same exposure time (6h) resulted in a significant (P<0.05) decrease in L. monocytogenes counts on frankfurters of approximately 2logCFUg(-1) after 14 days of storage as compared to the control. Both the above-mentioned films showed a similar effectiveness in reducing total aerobic bacterial populations as measured by total aerobic plate counts on frankfurters. For both films, total aerobic bacterial levels were significantly (P>0.05) reduced by approximately 3.3logCFUg(-1) after 14 days of storage as compared to control samples. Bacterial cellulose films were demonstrated in this study to have potential applicability as antimicrobial packaging films or inserts for processed meat products.

Structure of Acetobacter cellulose composites in the hydrated state.

The structure of composites produced by the bacterium Acetobacter xylinus have been studied in their natural, hydrated, state. Small-angle X-ray diffraction and environmental scanning electron microscopy has shown that the ribbons have a width of 500 A and contain smaller semi-crystalline cellulose microfibrils with an essentially rectangular cross-section of approximately 10 x 160 A(2). Incubation of Acetobacter in xyloglucan or pectin results in no changes in the size of either the microfibrils or the ribbons. Changes in the cellulose crystals are seen upon dehydration of the material, resulting in either a reduction in crystal size or an increase in crystal disorder.

The phase transformations in starch during gelatinisation: a liquid crystalline approach.

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.

Probing expansin action using cellulose/hemicellulose composites.

Cellulose-based composite materials containing xyloglucans or mannan-based polysaccharides have been shown to possess organisational features with many characteristics similar to primary plant cell walls. We have tested the effects of a typical alpha-expansin (CsExp1) on these composites using two different mechanical assays. We show that CsExp1 induces very rapid extension in composites containing tamarind xyloglucan under constant load. In contrast, expansin treatment had no effect in constant load extension assays using cellulose-only materials or in those carried out on composites containing glucomannan or galactomannan. We show that the effect of expansins is much smaller on composites made with short chain length xyloglucans than on those containing longer chains. In uniaxial extension tests we found that expansin could double the total extension (before failure) in xyloglucan composites and that the effects were again lower in composites containing shorter xyloglucans. We found no effect of expansin on uniaxial extensions with glucomannan or galactomannan. However, a significant effect of expansin on the uniaxial extension behaviour of cellulose-only material was observed. These experiments suggest that the target of CsExp1 in cell walls is probably the cellulose xyloglucan matrix, but that other (1-4) beta-glucan to (1-4) beta-glucan hydrogen bonded contacts can also serve as substrates.

Production of very-high-amylose potato starch by inhibition of SBE A and B.

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.

Temporal and spatial regulation of pectic (1-->4)-beta-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties.

Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.

Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis.

Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1-->4)-beta-D-mannan synthase and a UDP-galactose dependent (1-->6)-alpha-D-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1-->4)-linked D-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose-substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan alpha-galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed.

A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A.

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.

A new family of oligosaccharides from the xyloglucan of Hymenaea courbaril L. (Leguminosae) cotyledons.

The xyloglucan from cotyledons of Hymenaea courbaril was hydrolysed with endo-(1,4)-beta-D-glucanase (cellulase) and analysed by TLC and HPAEC. The limit digest was different from those obtained from xyloglucans of Tamarindus indica and Copaifera langsdorffii. On treatment with nasturtium beta-galactosidase, two main oligosaccharides were detected by TLC and HPAEC. Using a process of enzymatic sequencing involving alternate treatments with a pure xyloglucan oligosaccharide-specific alpha-xylosidase, and a pure beta-glucosidase, both from nasturtium, their structures were deduced to be XXXG and a new oligosaccharide XXXXG. These structures were confirmed by 1H NMR. The relative proportions of XXXG and XXXXG indicate that approximately half of the subunits in Hymenaea xyloglucan are based on the new oligosaccharides. In the native polymer the XXXXG subunits are likely to carry galactosyl substituents in varying proportions, since cellulase hydrolysates contained many bands which were converted to XXXXG on hydrolysis with nasturtium beta-galactosidase. Although no comparative studies on the physico-chemical properties of Hymenaea courbaril xyloglucan have yet been performed, our results indicate that this polymer is less interactive with iodine when compared with T. indica and C. langsdorffii xyloglucans, suggesting that changes in conformation may occur due to the presence of XXXXG.

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1-->4)-beta-D-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds.

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(1-->4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered -3, -2, +2 and +3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. [formula: see text] We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose-substitution at glucose residues -4, -2, +1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.