Galactan Side Chains Research Articles

Associating structural characteristics to immunomodulating properties of carrot rhamnogalacturonan-I fractions

Carrot rhamnogalacturonan-I (cRG-I) is a polydisperse polysaccharide with molecular weights of 7–250 kDa. Using size exclusion chromatography cRG-I was fractionated and pooled in fractions (PF1–6). All fractions contained the same RG-I monosaccharides and similar glycosidic linkages although in varying relative amounts. The main differences were in rhamnose substitution, arabinan- and galactan side chain length and in levels of acetylation and methyl esterification. Atomic force microscopy showed either spheric or elongated structures for cRG-I and its derived fractions. To gain insight in the structure-function relationship of cRG-I, the immunomodulatory effect of the six fractions and their saponified derivatives was assessed in vitro. All fractions, except PF2, dose-dependently stimulated TNFα, IL-6, IL-1β, IL-8 and IL-10 production in peripheral blood mononuclear cells (PBMCs) of three healthy donors. Cytokine levels were largely influenced by the Mw and degree of esterification of the individual fractions. Notably, the highest Mw fraction (100 kDa) displayed the most potent activity, which was strongly reduced after the removal of ester residues by saponification. In contrast, the 75 kDa Mw population (PF2) proved inactive while its saponified counterpart exhibited substantial immunomodulatory activity. This confirmed the role of ester residues on the immune profile of RG-I subpopulations.

Extensive Analysis of Mulberry (Morus rubra L.) Polysaccharides with Different Maturities by Using Two-Step Extraction and LC/QqQ-MS.

A primary challenge of polysaccharide analysis is the need for comprehensive extraction and characterization methods. In this study, mulberry polysaccharides at different maturities were fully extracted through a two-step process involving ethylenediaminetetraacetic acid (EDTA) and sodium hydroxide (NaOH), and their structures were determined by a combination analysis of monosaccharides and glycosidic linkages based on liquid chromatography triple quadrupole mass spectrometry (LC/QqQ-MS). The results indicate mulberry polysaccharides mainly contain highly branched pectic polysaccharides, (1,3,6)-linked glucan, xylan, and xyloglucan, but the content of different portions varies at different maturity stages. HG decreases from 19.12 and 19.14% (green mulberry) to 9.80 and 6.08% (red mulberry) but increases to 17.83 and 11.83% as mulberry transitioned from red to black. In contrast, the contents of glucan showed opposite trends. When mulberry turns red to black, the RG-I arabinan chains decrease from 47.75 and 28.86% to 13.16 and 12.72%, while the galactan side chains increase from 1.18 and 1.91 to 8.3 and 6.49%, xylan and xyloglucan show an increase in content. Overall, the two-step extraction combined with LC/QqQ-MS provides a new strategy for extensive analysis of complex plant polysaccharides.

Rhamnogalacturonan I with β-(1,4)-Galactan Side Chains as an Ever-Present Component of Tertiary Cell Wall of Plant Fibers.

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.

Analysis of pectin-cellulose interaction in cell wall of lotus rhizome with assistance of ball-milling and galactosidase

The current study sought to depict the structural feature of polysaccharides extracted from Na2CO3 unextractable fraction (LUN) of lotus rhizome using galactosidase with assistance of ball milling. The extracted polysaccharides were a complex of cellulose microfibrils and the RG-I structural domain of pectin, and the top three monosaccharides were glucose, galactose and galactose uronic acid, which allowed to tune the properties of the enzyme-hydrolyzed polysaccharide from LUN after 15 and 45 min of ball milling. The data of XRD revealed that pectin has a masking effect on the diffraction peaks of cellulose components. The removing of the polysaccharides could increase the degree of crystallinity and the pectin-cellulose interaction mainly occured through the galactan side chain was speculated. Textural characterization by SEM exhibited a cross-linked rod-like structure, which is similar to the structure of cellulose microfibrils. The morphological analysis of AFM revealed that L15-P (enzyme-hydrolyzed polysaccharide from LUN after 15 min of ball milling) contained relatively ordered and uniform network structures. Overall, the present study provides an important insight into cell wall of lotus rhizome matrix polysaccharide.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Immunohistochemical dynamics of cell wall matrix polymers during tomato autograft healing

A large part of the production of tomato plants is grafted. Although it has recently been described that cell walls play an important role in tomato graft healing, the spatiotemporal dynamics of cell wall changes in this critical process remains largely unknown. The aim of this work was to immunolocalize changes in the major cell wall matrix components of autograft union tissues throughout the course of healing, from 1 to 20 days after grafting (DAG). Homogalacturonan was de novo synthetized and deposited in the cut edges, displaying the low methyl-esterified homogalacturonan a stronger labelling. Labelling of galactan side chains of rhamnogalacturonan increased until 8 DAG, although remarkably a set of cells at the graft union did not show labelling for this epitope. Changes in xylan immunolocalization were associated to the xylem vasculature development throughout, while those of xyloglucan revealed early synthesis at the cut edges. Arabinogalactan proteins increased up to 8 DAG and showed scion-rootstock asymmetry, with a higher extent in the scion. The combination of these changes appears to be related with the success of the autograft, specifically facilitating the adhesion phase between scion-rootstock tissues. This knowledge paves the way for improved grafting using methods that facilitate appropriate changes in the time and space dynamics of these cell wall compounds.

Structural and biochemical insight into a modular β-1,4-galactan synthase in plants.

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the β-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.

A novel pectic polysaccharide-based hydrogel derived from okra (Abelmoschus esculentus L. Moench) for chronic diabetic wound healing

Hydrogels based on natural polysaccharides represent a growing group of suitable biomaterials for the elaboration of effective wound healing dressings, especially for the treatment of chronic wounds. This work was intended to prepare a polysaccharide-based hydrogel for diabetic wound healing which would help maintain the well-being of diabetes and improve their quality of life. For this purpose, a pectic polysaccharide (OPS) was extracted and purified, for the first time, from Tunisian okra pods and its physicochemical and rheological features, antioxidant and in vivo and in vitro wound healing activities were investigated. OPS, an acidic polysaccharide with a molecular weight of 3.28 × 106 Da and a polydispersity index of 1.03, was mainly composed of galactose (24.45 %), galacturonic acid (24.6 %) and rhamnose (18.25 %). Combined with FT-IR and NMR analyses, it consisted of a pectic rhamnogalacturonan I (RG-I) structure with galactan side chains. The OPS demonstrated antioxidant potential, gelling ability, cytocompatibility properties, non-cytotoxicity and cell migration and proliferation promoting activities, which met the requirements for wound dressings. Then, the in vivocutaneous wound healing effect of OPS-based hydrogel was investigated using an alloxan-induced diabetic rat model, and results showed that it significantly accelerated the wound healing process by acting in the acceleration of the recovery of the dermis and inducing moreblood vessels formationand tissue granulation.Overall, these results provide new insights into the development of a promising and effective okra pectin-based hydrogel for the treatment of chronic diabetic wounds.

Protective effects of six different pectic polysaccharides on DSS-induced IBD in mice

Administration of pectic polysaccharides is a therapeutic strategy in the management of gut inflammation. However, the knowledge about the effects of different dietary sources and structure are missing. This study explored the protective effects of six different pectic polysaccharides from Goji berry (G), dragon fruit (D), lemon (L), pomelo (P), potato (T) and raspberry (R) in dextran sulfate sodium (DSS)-induced colitis mice. D mainly contained rhamnogalacturonan (RG) backbone; L had high amount of arabinan sidechains; P was a high-esterified homogalacturonan (HG) pectic polysaccharide; the primary domain in T was galactan sidechains; R and G had the composition of arabinan: galactan: RG: HG = 19: 28: 15: 22 and 36: 17: 21: 14. Our results suggested that G and R were the most effective in alleviating colitis via alleviating tissue damage, promoting the index of immune organs, suppressing the production of TNF-α, IL-1β and IL-17, improving antioxidant status and promoting the total amount of SCFAs. Similarly, the administration of L alleviated colitis mainly by increasing the fermentation products without changes in the composition pattern of SCFAs. The secretion of IL-6 might be suppressed by pectic polysaccharides containing high amount of arabinan sidechains, while anti-inflammatory cytokines and intestinal barrier function tend to be regulated by pectic polysaccharides rich in galactan. In conclusion, pectic polysaccharide containing diversified structural domains (homogeneous proportion of HG, RG, arabinan, and galactan) might be the most effective in alleviating gut inflammation. This unprecedented report revealed the anti-inflammatory effects of different pectic polysaccharides, which was critical for the reasonable selection of dietary fiber to prevent gut inflammation.

Consistent Prebiotic Effects of Carrot RG-I on the Gut Microbiota of Four Human Adult Donors in the SHIME® Model despite Baseline Individual Variability.

The human gut microbiome is currently recognized to play a vital role in human biology and development, with diet as a major modulator. Therefore, novel indigestible polysaccharides that confer a health benefit upon their fermentation by the microbiome are under investigation. Based on the recently demonstrated prebiotic potential of a carrot-derived pectin extract enriched for rhamnogalacturonan I (cRG-I), the current study aimed to assess the impact of cRG-I upon repeated administration using the M-SHIME technology (3 weeks at 3g cRG-I/d). Consistent effects across four simulated adult donors included enhanced levels of acetate (+21.1 mM), propionate (+17.6 mM), and to a lesser extent butyrate (+4.1 mM), coinciding with a marked increase of OTUs related to Bacteroides dorei and Prevotella species with versatile enzymatic potential likely allowing them to serve as primary degraders of cRG-I. These Bacteroidetes members are able to produce succinate, explaining the consistent increase of an OTU related to the succinate-converting Phascolarctobacterium faecium (+0.47 log10(cells/mL)). While the Bifidobacteriaceae family remained unaffected, a specific OTU related to Bifidobacterium longum increased significantly upon cRG-I treatment (+1.32 log10(cells/mL)). Additional monoculture experiments suggested that Bifidobacterium species are unable to ferment cRG-I structures as such and that B. longum probably feeds on arabinan and galactan side chains of cRG-I, released by aforementioned Bacteroidetes members. Overall, this study confirms the prebiotic potential of cRG-I and additionally highlights the marked consistency of the microbial changes observed across simulated subjects, suggesting the involvement of a specialized consortium in cRG-I fermentation by the human gut microbiome.

Dynamic changes of pectin epitopes and daylily tepals during flower opening and senescence of Hemerocallis citrina

Ephemeral flower deaths occur within 24 h of flowering in the genus Hemerocallis. The senescence of plant organs is owing to the loss of cell wall cohesion. Pectin is the most structurally complex plant cell wall polysaccharide and has an important role in cell-to-cell adhesion. In this study, we used histological stains, immunolocalization and pectin characterization analyses to examine the dynamic changes in pectin in Hemerocallis citrina ‘Datonghuanghua’ tepals at eight stages (S1–S8). The pectin-specific antibodies JIM5 and JIM7, which hybridize to differently esterified regions of the homogalacturonan (HG) backbone, showed that demethylesterification of HG-type pectins was evident after the outer tepals had opened. Before flower opening, these two antibodies labelled tissue continuously across the cell wall, and the cells were round and tightly packed. The labeling of highly esterified epitopes recognized by JIM7 became deformed when the outer tepals opened. However, 12 h after blooming, low-and high-esterified epitope signals were disrupted near the vascular cylinder, and air space then appeared until these signals disappeared. labeling with the antibody LM5, which is specific for the (1 → 4)-β galactan side chains of rhamnogalacturonan-I, was disrupted near the vascular cylinder at the same stage. Moreover, the contents of neutral sugars in pectin analyses showed that galactose decreased significantly at 12 h after blooming. Three enzymes were involved in pectin remodeling at different developmental phases. The activities of pectin methylesterase continued to increase after the outer tepals opened and reached a significant peak at 12 h after blooming. The activities of polygalacturonase and pectate lyase also increased, but the difference was not significant. Our findings revealed that demethylesterification of HG-type pectins was evident after the outer tepals opened, and this remodeling greatly facilitated their degradation by pectic enzymes. The loss of cell wall material and cohesion that accompanied the activities of pectic enzymes was associated with senescence. Therefore, remodeling of the pectin in cell walls of the tepals is necessary for the occurrence of senescence.

Cell Wall Layer Induced in Xylem Fibers of Flax Upon Gravistimulation Is Similar to Constitutively Formed Cell Walls of Bast Fibers.

In the fibers of many plant species after the formation of secondary cell walls, cellulose-enriched cell wall layers (often named G-layers or tertiary cell walls) are deposited which are important in many physiological situations. Flax (Linum usitatissimum L.) phloem fibers constitutively develop tertiary cell walls during normal plant growth. During the gravitropic response after plant inclination, the deposition of a cellulose-enriched cell wall layer is induced in xylem fibers on one side of the stem, providing a system similar to that of tension wood in angiosperm trees. Atomic force microscopy (AFM), immunochemistry, and transcriptomic analyses demonstrated that the G-layer induced in flax xylem fibers was similar to the constitutively formed tertiary cell wall of bast (phloem) fibers but different from the secondary cell wall. The tertiary cell walls, independent of tissue of origin and inducibility, were twice as stiff as the secondary cell walls. In the gravitropic response, the tertiary cell wall deposition rate in xylem was higher than that of the secondary cell wall. Rhamnogalacturonan I (RG-I) with galactan side chains was a prominent component in cellulose-rich layers of both phloem and xylem flax fibers. Transcriptomic events underlying G-layer deposition in phloem and xylem fibers had much in common. At the induction of tertiary cell wall deposition, several genes for rhamnosyltransferases of the GT106 family were activated in xylem samples. The same genes were expressed in the isolated phloem fibers depositing the tertiary cell wall. The comparison of transcriptomes in fibers with both inducible and constitutive tertiary cell wall deposition and xylem tissues that formed the secondary cell walls is an effective system that revealed important molecular players involved in the formation of cellulose-enriched cell walls.

Modelling the Extraction of Pectin towards the Valorisation of Watermelon Rind Waste.

Watermelon is the second largest fruit crop worldwide, with great potential to valorise its rind waste. An experimental design was used to model how extraction parameters (temperature, pH, and time) impact on the efficiency of the process, purity, esterification degree, monosaccharide composition and molar mass of watermelon rind pectin (WRP), with an insight on changes in their structural properties (linearity, branching degree and extraction severity). The models for all responses were accurately fitted (R2 > 90%, lack of fit p ≥ 0.05) and experimentally validated. At optimum yield conditions, WRP yield (13.4%), purity (540 µg/g galacturonic acid) and molar mass (106.1 kDa) were comparable to traditional pectin sources but showed a higher branching degree with longer galactan side chains and a higher protein interaction. Harsher conditions (pH 1) generated purer homogalacturonan fractions with average molar masses (80 kDa) at the expense of yield, while mild extraction conditions (pH ≥ 2) produced highly branched entangled pectin structures. This study underlines novel compositional features in WRP and the possibility of producing novel customized pectin ingredients with a wider potential application scope depending on the targeted structure.

Pectic polysaccharides from Radix Sophorae Tonkinensis exhibit significant antioxidant effects

Two pectic polysaccharides (WRSP-A2b and WRSP-A3a) have been obtained from Radix Sophorae Tonkinensis and comparatively investigated in terms of their physical properties and antioxidant activities. Monosaccharide composition, FT-IR, NMR and enzymatic analyses indicate that both WRSP-A2b (13.6 kDa) and WRSP-A3a (44.6 kDa) consist of homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains, with mass ratios of 0.9:1.8:1 and 2.3:2.9:1, respectively. The RG-I domains were further purified and characterized. Results show that WRSP-A2b contains a highly branched RG-I domain, primarily substituted with α-(1→5)-linked arabinans, whereas WRSP-A3a contains a small branched RG-I domain mainly composed of β-(1→4)-linked galactan side chains. WRSP-A3a exhibits stronger antioxidant activity in scavenging different radicals than WRSP-A2b, a finding that may be due to its higher content of GalA residues and HG domains. Our results provide useful information for screening natural polysaccharide-based antioxidants from Radix Sophorae Tonkinensis.

Structural characteristics of a highly branched and acetylated pectin from Portulaca oleracea L.

The water-extracted polysaccharide from Portulaca oleracea L. mainly consisted of two pectic polysaccharides designated as POWP-H and POWP-L. The POWP-L was rich in linear homogalacturonan with GalA content of 77.6%, while POWP-H might be a rhamnogalacturonan I (RG-I) pectin composed of Rha (15.7%), Ara (31.7%), Gal (42.5%) and GalA (10.1%). The structure of POWP-H was elucidated by partial degradation, methylation analysis combined with GC-MS method and NMR spectroscopy. It had a weight-average molecular weight of 1.2 × 106 g/mol and high degree of branch of 0.57. The presence of abundant arabinogalactan-II and certain galactan side chains in POWP-H was verified by the T-Araf (17.4%), 1,3,6-Galp (11.8%), 1,3-Galp (8.1%), T-Galp (13.4%) and 1,4-Galp (14.3%). The evidence for the RG-I backbone of POWP-H was demonstrated by the structure analysis of its degraded product POWP-H-H, which had the backbone of →4)-α-GalpA-(1 → 2)-α-Rhap-(1 → 4)-α-GalpA-(1 → 2,4)-α-Rhap-(1→ substituted by short side chain of β-Galp-(1 → 4)-β-Galp-(1→ at O-4 position of →2,4)-α-Rhap-(1 → . Moreover, the POWP-H-H was mainly O-acetylated at O-3 position of α-Rhap with 24.8% acetylation degree. In conclusion, POWP-H was mainly the highly branched and acetylated RG-I pectin with relative short RG-I backbone and abundant arabinogalactan II and certain galactan side chains. Besides, it adopted a flexible chain conformation in 0.1 M NaNO3 solution.

Effect of surfactant-induced competitive displacement of whey protein conjugated to acid- or alkali-extracted potato pectin on emulsion stability

Covalently linked protein–polysaccharide conjugate pre-adsorbed to the interface often shows more enhanced resistance to displacement induced by competitively adsorbing small molecule surfactant than the pure protein component. The objective of this study was to investigate the effect of surfactant-induced competitive displacement of whey protein–potato pectin conjugate pre-adsorbed to oil droplet surfaces on the stability of the oil-in-water emulsion. Potato pectin extracted in either an acidic or alkaline condition was conjugated to bovine milk whey protein using the Maillard reaction. The formation of covalent linkage between the protein and pectin moieties was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Atomic force microscopy studies of the interfacial films of the Maillard conjugates formed on flat water surfaces revealed that the alkali-extracted potato pectin provided more enhanced resistance to surfactant-induced competitive displacement from the air–water interface than the acid-extracted potato pectin. These results indicate that galactan side chains having an ability to self-assemble and form network structures had been preserved to a larger degree during the alkali-extraction. Furthermore, the alkali-extracted potato pectin prevented coalescence-induced increases in the oil droplet diameter in the oil-in-water emulsions prepared using the Maillard conjugate as the emulsifier upon the addition of up to 38.7 mM of the Tween 20 surfactant while maintaining relatively high levels of the surface load of protein (>0.8 mg/m2). The present results demonstrate that the molecular structure of the polysaccharide moiety of protein–polysaccharide conjugate has significant impacts on its resistance to competitive displacement and emulsion stability.

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Extraction and isolation of pectin rich in homogalacturonan domains from two cultivars of hawthorn berry (Crataegus pinnatifida)

Hawthorn berries present high pectin content with good ability to form and stabilize oil-in-water emulsions. However, successful applications are determinant of high yield and purity as well as good surfactant properties based on their molecular structure. In this work, pectins from one of the most common Crataegus pinnatifida cultivars (Dajinxing, DA), and from a less known cultivar gaining popularity (Mianqiu, MI), were extracted and isolated. They were comparatively investigated with two commercial pectins from citrus peel [> 67.9% degree of methyl-esterification (DM) and >78% galacturonic acid (GalA)] and analyzed for their yield, molecular weight (HPSEC-MALS-RI), monomer composition and glycosyl linkage analysis (GC-MS), DM (titration, FTIR and 1H NMR), and emulsifying performance. MI showed a 2-fold greater pectin yield (in terms of GalA) than DA (61.2% vs. 38.3%). Thus, hot acid extraction had to be followed by an enzymatic step to remove starch molecules and obtain purified DA and MI pectins (>86% GalA), which also resulted in pectin demethoxylation (DM <38%). Remarkably, purified hawthorn pectins presented lower molecular weight than commercial citrus pectins (98–126 kDa vs 164–235 kDa) together with a higher proportion of homogalacturonan (HG) domain and a significantly greater emulsifying activity and stability. Specifically, MI showed a higher HG proportion and a less branched rhamnogalacturonan-I (RG-I) domain [Branching calculated as (Arabinose + Galactose)/Rhamnose = 2.2] than DA (Branching = 3.1), with significantly shorter arabinan and galactan side chains (evidenced by more terminal arabinose and galactose). The yield and structure of MI pectin strongly indicates that MI berry is an excellent pectin source with distinguished functionality.

Structural insights in interactions between RNase from Bacillus Intermedius and rhamnogalacturonan I from potato

Being biocompatible and biodegradable polymers, polysaccharides present a perspective material for drug delivery systems. This study aimed at unraveling the molecular details of interactions between rhamnogalacturonan I, brunched with galactan side chains, and RNase from Bacillus Intermedius, binase. FTIR- and NMR-spectroscopic analyses showed that binase interacts with side chains of the polysaccharide. In complexes with polysaccharide, the protein retains its native structure. The 2D-NMR techniques revealed eight protein residues responsive to polysaccharide binding. Further, computer simulations were carried out to provide the atomistic details of binase–polysaccharide complexes. Both blind and knowledge-based docking procedures elucidate the existence of epitopes on the binase surface with the preferential binding of galactan fragments. The refinement of these complexes by molecular dynamics simulations confirmed stable protein–polysaccharide interactions. The results of this study strengthen the knowledge on non-specific protein-carbohydrate interactions and outline the rhamnogalacturonan I as a possible matrix material for protein delivery systems.

Structure-activity relationship of Citrus segment membrane RG-I pectin against Galectin-3: The galactan is not the only important factor

Rhamnogalacturonan I (RG-I) pectin are regarded as strong galectin-3 (Gal-3) antagonist because of galactan sidechains. The present study focused on discussing the effects of more structural regions in pectin on the anti-Gal-3 activity. The water-soluble pectin (WSP) recovered from citrus canning processing water was categorized as RG-I pectin. The controlled enzymatic hydrolysis was employed to sequentially remove the α-1,5-arabinan, homogalaturonan and β-1,4-galactan in WSP. The Gal-3-binding affinity KD (kd/ka) of WSP and debranched pectins were calculated to be 0.32 μM, 0.48 μM, 0.56 μM and 1.93 μM. Moreover, based on the more sensitive cell line (MCF-7) model, the IC30 value of WSP was lower than these of modified pectins, indicating decreased anti-Gal-3 activity. Our results suggested that the total amount of neutral sugar sidechains, the length of arabinan and cooperation between HG and RG-I played important roles in the anti-Gal-3 activity of WSP, not the Gal/Ara ratio or RG-I/HG ratio. These results provided a new insight into structure-activity relationship of citrus segment membrane RG-I as a galectin-3 antagonist and a new functional food.