Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

Pectin and its derivatives have been shown to modulate immune signaling as well as gut microbiota in preclinical studies, which may constitute the mechanisms by which supplementation of specific pectic polysaccharides confers protection against viral respiratory infections. In a double-blind, placebo-controlled rhinovirus (RV16) challenge study, healthy volunteers were randomized to consume placebo (0.0 g/day) (N = 46), low-dose (0.3 g/day) (N = 49) or high-dose (1.5 g/day) (N = 51) of carrot derived rhamnogalacturonan-I (cRG-I) for eight weeks and they were subsequently challenged with RV-16. Here, the effect of 8-week cRG-I supplementation on the gut microbiota was studied. While the overall gut microbiota composition in the population was generally unaltered by this very low dose of fibre, the relative abundance of Bifidobacterium spp. (mainly B. adolescentis and B. longum) was significantly increased by both doses of cRG-1. Moreover, daily supplementation of cRG-I led to a dose-dependent reduction in inter- and intra-individual microbiota heterogeneity, suggesting a stabilizing effect on the gut microbiota. The severity of respiratory symptoms did not directly correlate with the cRG-I-induced microbial changes, but several dominant groups of the Ruminococcaceae family and microbiota richness were positively associated with a reduced and hence desired post-infection response. Thus, the present results on the modulation of the gut microbiota composition support the previously demonstrated immunomodulatory and protective effect of cRG-I during a common cold infection.

UNASSIGNED: Panax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear.
UNASSIGNED: P. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis.
UNASSIGNED: Our ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar KD values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function.
UNASSIGNED: P. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

Pectin, a complex polysaccharide found in plant cell walls, plays a crucial role in various industries due to its functional properties. The diluted alkali-soluble pectin (DASP) fractions that result from the stepwise extraction of apples and carrots were studied to evaluate their structural and rheological properties. Homogalacturonan and rhamnogalacturonan I, in different proportions, were the main pectin domains that composed DASP from both materials. Atomic force microscopy revealed that the molecules of apple DASP were longer and more branched. A persistence length greater than 40 nm indicated that the pectin molecules deposited on mica behaved as stiff molecules. The weight-averaged molar mass was similar for both samples. Intrinsic viscosity values of 194.91 mL·g-1 and 186.79 mL·g-1 were obtained for apple and carrot DASP, respectively. Rheological measurements showed greater structural strength for apple-extracted pectin, whereas carrot pectin was characterized by a higher linear viscoelasticity limit. This comparison showed that the pectin fractions extracted by diluted alkali are structurally different and have different rheological properties depending on their botanical origin. The acquired insights can enhance the customized use of pectin residue and support further investigations in industries relying on pectin applications.

Glycosylation is a method that enhances the functional properties of proteins by covalently attaching sugars to them. This study aimed at preparing three conjugates (WP-HG, WP-SBP, and WP-RGI) by dry heating method to research the influence of different pectin structures on the functional properties of WP and characterize properties and structures of these conjugates. The research results manifested that the degree of glycosylation (DG) of HG, SBP and RGI were 13.13 % ± 0.07 %, 23.27 % ± 0.3 % and 36.39 % ± 0.3 % respectively, suggesting that the increase of the number of branch chains promoted the glycosylation reaction. The formation of the conjugate was identified by the FT-IR spectroscopy technique. And SEM showed that WP could covalently bind to pectin, resulting in a smoother and denser surface of the conjugates. The circular dichroism analysis exhibited that the glycosylation reaction altered the secondary structure of WP and decreased the α-Helix content. This structural change in the protein spatial conformation led to a decrease in the hydrophobicity of protein surface. But the addition of pectin further regulated the hydrophilic-hydrophobic ratio on the surface of the protein, thus improving the emulsification properties of WP. In addition, the glycosylation could improve the stability of the emulsion, giving it a smaller droplet size, higher Zeta-potential and more stable properties. In a word, this study pointed out the direction for the application of different pectin structures in the development of functional properties of glycosylation products in food ingredients.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the \"hairy\" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

The cellulose-enriched tertiary cell walls present in many plant fibers have specific composition, architecture, machinery of formation, and function. To better understand the mechanisms underlying their mode of action and to reveal the peculiarities of fibers from different plant species, it is necessary to more deeply characterize the major components. Next to overwhelming cellulose, rhamnogalacturonan I (RG-I) is considered to be the key polymer of the tertiary cell wall; however, it has been isolated and biochemically characterized in very few plant species. Here, we add RG-I to the list from the phloem fibers of the Phaseolus vulgaris stem that was isolated and analyzed by nuclear magnetic resonance (NMR), dynamic light scattering, and immunolabeling, both within tissue and as an isolated polymer. Additionally, fibers with tertiary cell walls from nine species of dicotyledonous plants from the orders Malphigiales, Fabales, and Rosales were labeled with RG-I-related antibodies to check the presence of the polymer and compare the in situ presentation of its backbone and side chains. The obtained results confirm that RG-I is an obligatory polymer of the tertiary cell wall. However, there are differences in the structure of this polymer from various plant sources, and these peculiarities may be taxonomically related.

The human gut microbiota is characterized by large interpersonal differences, which are not only linked to health and disease but also determine the outcome of nutritional interventions. In line with the growing interest for developing targeted gut microbiota modulators, the selectivity of a carrot-derived rhamnogalacturonan I (cRG-I) was compared to substrates with demonstrated low (inulin, IN) and high selectivity (xanthan, XA), at a human equivalent dose (HED) of 1.5 g/d. The high throughput of the ex vivo SIFR® technology, validated to generate predictive insights for clinical findings, enabled the inclusion of 24 human adults. Such an unprecedented high number of samples in the context of in vitro gut microbiota modelling allowed a coverage of clinically relevant interpersonal differences in gut microbiota composition and function. A key finding was that cRG-I supplementation (already at an HED of 0.3 g/d) lowered interpersonal compositional differences due to the selective stimulation of taxa that were consistently present among human adults, including OTUs related to Bacteroides dorei/vulgatus and Bifidobacterium longum (suspected keystone species), Bacteroides thetaiotaomicron, Bifidobacterium adolescentis and butyrate-producing taxa such as Blautia sp., Anaerobutyricum hallii, and Faecalibacterium prausnitzii. In contrast, both IN and XA treatments increased interpersonal compositional differences. For IN, this followed from its low specificity. For XA, it was rather the extremely high selectivity of XA fermentation that caused large differences between 15 responders and 9 nonresponders, caused by the presence/absence of highly specific XA-fermenting taxa. While all test compounds significantly enhanced acetate, propionate, butyrate, and gas production, cRG-I resulted in a significantly higher acetate (+40%), propionate (+22%), yet a lower gas production (-44%) compared to IN. cRG-I could thus result in overall more robust beneficial effects, while also being better tolerated. Moreover, owing to its remarkable homogenization effect on microbial composition and metabolite production, cRG-I could lead to more predictable outcomes compared to substrates that are less specific or overly specific.

This study is the first to extract and characterize pectin from citrus physiological premature fruit drop. The extraction yield of pectin reached 4.4 % by acid hydrolysis method. The degree of methoxy-esterification (DM) of citrus physiological premature fruit drop pectin (CPDP) was 15.27 %, indicating it was low-methoxylated pectin (LMP). The monosaccharide composition and molar mass test results showed CPDP was a highly branched macromolecular polysaccharide (β: 0.02, Mw: 2.006 × 105 g/mol) with rich rhamnogalacturonan I domain (50.40 %) and long arabinose and galactose side chain (32.02 %). Based on the fact that CPDP is LMP, Ca2+ was used to induce CPDP to form gels. Textural and rheological tests showed that the gel strength and storage modulus of CPDP were higher than commercial citrus pectin (CP) used in this paper due to the lower DM and rich neutral sugar side chains of CPDP. Scanning electron microscope (SEM) results showed CPDP had stable gel network structure.

Therapy of colorectal cancer with protein drugs, including targeted therapy using monoclonal antibodies, requires the preservation of the drug\'s structure and activity in the gastrointestinal tract or bloodstream. Here, we confirmed experimentally the fundamental possibility of creating composite protein-polysaccharide hydrogels based on non-degrading rhamnogalacturonan I (RG) and fibrin as a delivery vehicle for antitumor RNase binase. The method is based on enzymatic polymerization of fibrin in the presence of RG with the inclusion of liposomes, containing an encapsulated enzyme drug, into the gel network. The proposed method for fabricating a gel matrix does not require the use of cytotoxic chemical cross-linking agents and divalent cations, and contains completely biocompatible and biodegradable components. The process proceeds under physiological conditions, excluding the effect of high temperatures, organic solvents and ultrasound on protein components. Immobilization of therapeutic enzyme binase in the carrier matrix by encapsulating it in liposomes made from uncharged lipid made it possible to achieve its prolonged release with preservation of activity for a long time. The release time of binase from the composite carrier can be regulated by variation of the fibrin and RG concentration.