Structural variety of glucans from Ganoderma lucidum fruiting bodies.

Ganoderma lucidum, widely used in traditional medicine, has several biological properties. Polysaccharides, mainly glucans, are known as one of its main bioactive compounds. Consequently, the achievement and chemical investigation of such molecules are of pharmaceutical interest. Herein, we obtained water-insoluble and water-soluble polysaccharides from G. lucidum by alkaline extraction. Fractionation process yielded three fractions (GLC-1, GLC-2, and GLC-3). All samples showed to be composed mainly of glucans. GLC-1 is a linear (1 â†’ 3)-linked ß-glucan; GLC-2 is a mixture of three different linear polysaccharides: (1 â†’ 3)-ß-glucan, (1 â†’ 3)-α-glucan, and (1 â†’ 4)-α-mannan; while GLC-3 is a branched ß-glucan with a (1 â†’ 4)-linked main chain, which is branched at O-3 or O-6 by (1 â†’ 3)- or (1 â†’ 6)-linked side chains. This research reports the variability of glucans in Ganoderma lucidum fruiting bodies and applicable methodologies to obtain such molecules. These polysaccharides can be further applied in biological studies aiming to investigate how their chemical differences may affect their biological properties.

Circular economyeast: Saccharomyces cerevisiae as a sustainable source of glucans and its safety for skincare application.

Glucans, a polysaccharide naturally present in the yeast cell wall that can be obtained from side streams generated during the fermentation process, have gained increasing attention for their potential as a skin ingredient. Therefore, this study focused on the extraction method to isolate and purify water-insoluble glucans from two different Saccharomyces cerevisiae strains: an engineered strain obtained from spent yeast in an industrial fermentation process and a wild strain produced through lab-scale fermentation. Two water-insoluble extracts with a high glucose content (> 90 %) were achieved and further subjected to a chemical modification using carboxymethylation to improve their water solubility. All the glucans' extracts, water-insoluble and carboxymethylated, were structurally and chemically characterized, showing almost no differences between both yeast-type strains. To ensure their safety for skin application, a broad safety assessment was undertaken, and no cytotoxic effect, immunomodulatory capacity (IL-6 and IL-8 regulation), genotoxicity, skin sensitization, and impact on the skin microbiota were observed. These findings highlight the potential of glucans derived from spent yeast as a sustainable and safe ingredient for cosmetic and skincare formulations, contributing to the sustainability and circular economy.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg-1) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2∼+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB α-glucanotransferases, which might facilitate the production of tailored α-glucans by enzyme engineering in future.

Rheology and gelation of aqueous carboxymethylated curdlan solution: Impact of the degree of substitution.

Curdlan is a unique (1,3)-ß-D-glucan with bioactivity and exceptional gelling properties. By chemical functionalization such as carboxymethylation, the physicochemical properties of curdlan can be significantly tailored. However, how the carboxymethylation extent of curdlan affects its rheology and gelation characteristics has yet to be fully understood. Herein, we investigated the impact of the degree of substitution (DS, ranging from 0.04 to 0.97) on the rheological and gelation behavior of carboxymethylated curdlan (CMCD). It was found that CMCD with DS below 0.20, resembling native curdlan, still retained its gelling capability. As the DS increased beyond 0.36, there was a significant increase in its water solubility instead of gelation, resulting in transparent solutions with steady/complex viscosities adhering to the Cox-Merz rule. Moreover, CMCD with high DS demonstrated the ability to undergo in-situ gelation in the presence of metal ions, attributed to the nonspecific electrostatic binding. Additionally, in vitro cytocompatibility testing showed positive compatibility across varying DS in CMCD. This research offers a holistic understanding of the viscosifying and gelling behaviors of CMCD with varying DS, thereby fostering their practical application as thickeners and gelling agents in fields ranging from food and biomedicine to cosmetics and beyond.

Preparation of ß-1,3-glucan mimics via modification of polymer backbone, and evaluation of cytokine production using the polymer library in immune activation.

ß-1,3-Glucans possess therapeutic potential owing to their ability to exhibit immunostimulating activity. ß-1,3-Glucans, isolated from various organisms, differ in their chemical structures, molecular weight, and branching degree, potentially forming particulate, helix, or random coil conformations in water. Therefore, this study used synthesized ß-1,3-glucan mimic polymers to investigate the difference in binding affinity for dectin-1 and induced cytokine productions based on polymer structures. The ß-1,3-glucan mimic polymers were synthesized using ß-1,3-glucan tetrasaccharyl monomer, with subsequent modifications to the polymer backbones through the introduction of hydrogen or a hydroxy group. Polymers with different structures in both ligands and polymer backbones were utilized to comprehensively investigate their binding affinity to dectin-1 and cytokine-inducing in macrophages. Hydroxylated polymers exhibited a high binding affinity for dectin-1, similar to that of schizophyllan, whereas the polymer composed of only saccharyl monomers did not bind to dectin-1. Further, when administered to macrophage RAW264 cells, polymers with branched and hydrophobic polymer backbones exhibited strong cytokine-inducing activities. Moreover, the results revealed that the essential factors for cytokine induction include the branches of ß-1,3-glucans, high (tens of thousands) molecular weights, and hydrophobicity. The results suggests that artificial polymers comprising these factors exhibit immunostimulating activity and could be developed as therapeutic agents.

Identification of a novel starch-converting GtfB enzyme from the Fructilactobacillus sanfranciscensis TMW11304 to reduce the viscoelasticity and retrogradation of tapioca starch.

Starch-converting α-glucanotransferases are efficient enzymatic toolkits for the biosynthesis of diverse α-glucans, which hold vast application potential in the food industry. In this work, we identified a novel GtfB protein from Fructilactobacillus sanfranciscensis TMW11304 (FsTMW11304 GtfB) in NCBI. Although this enzyme was highly conserved in motifs I-IV with those isomalto-maltopolysaccharides (IMMPs)-producing GtfB α-glucanotransferases, it possessed distinct deletions and mutations in two crucial loops shaping the active site. Hence, unlike those GtfB enzymes, FsTMW11304 GtfB not only exhibited excellent 4,6-α-glucanotransferase activity on amylose to generate atypically low-molecular-weight IMMPs with consecutive linear (α1 â†’ 6) linkages up to 48 %, but also held good capability towards branched substrates. Besides, compared with the control, the treatment by FsTMW11304 GtfB reduced the storage/loss modulus of granular and gelatinized tapioca starches (TS) by 12.0 %/17.9 % and 91.4 %/82.9 %, respectively, indicating that the rigidity of the gel structure was attenuated to different degrees in the two reaction systems. Furthermore, the setback viscosity observed in the gelatinized TS modified by FsTMW11304 GtfB was only 5 % of that observed in the control group, suggesting the short-term anti-retrogradation property has been substantially improved. Thus, FsTMW11304 GtfB represents a meaningful addition to the α-glucanotransferases in GH70 family, which expands the repertoire of diverse α-glucans synthesized from starch and facilitates the understanding of the structure-function relationship of the GtfB α-glucanotransferases.

Structure of rhamnoglucan, an unexpected alkali-stable polysaccharide extracted from Streptococcus mutans cell wall.

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.

Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Bifidobacterium Species and Its Physicochemical Properties.

α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from Bifidobacterium sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

(1 → 3),(1 → 6) and (1 → 3)-ß-D-glucan physico-chemical features drive their fermentation profile by the human gut microbiota.

Mushroom polysaccharides consist of a unique set of polymers that arrive intact in the human large intestine becoming available for fermentation by resident gut bacteria with potential benefits to the host. Here we have obtained four glucans from two mushrooms (Pholiota nameko and Pleurotus pulmonarius) under different extraction conditions and their fermentation profile by human gut bacteria in vitro was evaluated. These glucans were isolated and characterized as (1 â†’ 3),(1 â†’ 6)-ß-D-glucans varying in branching pattern and water-solubility. An aliquot of each (1 â†’ 3),(1 â†’ 6)-ß-D-glucan was subjected to controlled smith degradation process in order to obtain a linear (1 â†’ 3)-ß-D-glucan from each fraction. The four ß-D-glucans demonstrated different water solubilities and molar mass ranging from 2.2 × 105 g.mol-1 to 1.9 × 106 g.mol-1. In vitro fermentation of the glucans by human gut microbiota showed they induced different short chain fatty acid production (52.0-97.0 mM/50 mg carbohydrates), but an overall consistent high propionate amount (28.5-30.3 % of total short chain fatty acids produced). All glucans promoted Bacteroides uniformis, whereas Anaerostipes sp. and Bacteroides ovatus promotion was strongly driven by the ß-D-glucans solubility and/or branching pattern, highlighting the importance of ß-D-glucan discrete structures to their fermentation by the human gut microbiota.

Production, purification, and functional characterization of glucan exopolysaccharide produced by Enterococcus hirae strain OL616073 of fermented food origin.

Microbial exopolysaccharides (EPS) are high molecular weight polymeric substances with great diversity and variety of applications in the food and pharma industry. In this study, we report the extraction of an EPS from Enterococcus hirae OL616073 strain originally isolated from Indian fermented food and its purification by ion exchange and size exclusion chromatography for physical-functional analyses. The EPS showed two prominent fractions (EPS F1 and EPS F2) with molecular mass 7.7 × 104 and 6.5 × 104 Da respectively by gel permeation chromatography. These fractions were further characterized by FTIR, HPTLC, GC-MS, and NMR as a homopolysaccharide of glucose linked with α-(1 â†’ 6) and α-(1 â†’ 3) glycosidic linkages. The porous, spongy, granular morphology of EPS was observed under scanning electron microscopy. EPS has revealed strong physico-functional properties like water solubility index (76.75 %), water contact angle (65.74°), water activity (0.35), hygroscopicity (3.05 %), water holding capacity (296.19 %), oil holding capacity (379.91 %), foaming capacity (19.58 %), and emulsifying activity (EA1-72.22 %). Rheological analysis showed that aqueous solution of EPS exhibited a non-Newtonian fluid behavior and shear-thinning characteristics. Overall, EPS exhibits techno functional properties with potential applications as a functional biopolymer in food and pharma industry.

Recent innovations (2020-2023) in the approaches for the chemical functionalization of curdlan and pullulan: A mini-review.

Chemical modification represents a highly efficacious approach for enhancing the physicochemical characteristics and biological functionalities of natural polysaccharides. However, not all polysaccharides have considerable pharmacologic activity; so, appropriate chemical modification strategies can be selected in accordance with the distinct structural properties of polysaccharides to aid in improving and encouraging the presentation of their biological activities. Hence, there has been a growing interest in the chemical alteration of polysaccharides due to their various properties such as antioxidant, anticoagulant, antiviral, anticancer, biomedical, antibacterial, and immunomodulatory effects. This paper offers a comprehensive examination of recent scientific advancements produced over the past four years in the realm of unique chemical and functional modifications in curdlan and pullulan structures. This review aims to provide readers with an overview of the structural activity correlations observed in the backbone structures of curdlan and pullulan, as well as the diverse chemical modification processes employed for these polysaccharides. Additionally, the review aims to examine the effects of combining various bioactive molecules with chemically modified curdlan and pullulan and explore their potential applications in various important fields.

Exploring a GtfB-Type 4,6-α-Glucanotransferase to Synthesize the (α1 → 6) Linkages in Linear Chain and Branching Points from Amylose and Enhance the Functional Property of Granular Corn Starches.

Starch-converting α-glucanotransferases of glycoside hydrolase family 70 (GH70) are promising enzymatic tools for the production of diverse α-glucans with (potential) commercial applications in food and health and as biomaterials. In this study, a novel GtfB enzyme from Weissella confusa MBF8-1 was screened in the National Center for Biotechnology Information (NCBI) nonredundant protein database. The enzyme (named WcMBF8-1 GtfB) displayed high conservation in motifs I-IV with other GtfB enzymes but possessed unique variations in several substrate-binding residues. Structural characterizations of its α-glucan products revealed that WcMBF8-1 GtfB exhibited an atypical 4,6-α-glucanotransferase activity and was capable of catalyzing, by cleaving off (α1 → 4)-linkages in starch-like substrates and the synthesis of linear (α1 → 6) linkages and (α1 → 4,6) branching points. The product specificity enlarges the diversity of α-glucans and facilitates recognition of the determinants of the linkage specificity in GtfB enzymes. Furthermore, the contents of slowly digestible starch and resistant starch of granular corn starches, modified by WcMBF8-1 GtfB, increased by 6.7%, which suggested the potential value for the utilization of WcMBF8-1 GtfB to prepare "clean-label" starch ingredients with improved functional attributes.

Carboxyl-modified nanocellulose (cNC) enhances the stability of cNC/pullulan bio-nanocomposite hard capsule against moisture variation.

The quality of polysaccharide-based films and hard capsules is often affected by changes in relative humidity, manifesting as unstable water content, and changes in mechanical strength that make them brittle or soft. Herein, carboxyl-modified nanocellulose (cNC) was prepared and used as a new component to successfully improve the moisture resistance of cNC/pullulan/high-acyl gellan bio-nanocomposite hard capsules (NCPGs). Homogenously dispersed cNC in the pullulan/high-acyl gellan matrix could render the formation of more hydrogen bonds that provided additional water-binding sites and limited the free movement of pullulan and high-acyl gellan molecular chains within NCPGs. This contributed to a decreased amount of pooling adsorption water and an increased amount of Langmuir adsorption water in NCPGs, as compared to pullulan/high-acyl gellan hard capsules (PGs) without cNC. Therefore, the equilibrium moisture content (EMC) values of NCPGs decreased at 83 % relative humidity and increased at 23 % relative humidity compared to those of PGs. Together with enhanced mechanical and barrier properties, NCPGs effectively protected encapsulated amoxicillin and probiotic powder from changes in the outside humidity. Additionally, NCPGs exhibited faster drug release. This study presents a new mechanism and strategy for fabricating films and hard capsules with enhanced stability against moisture variation.

Magnetic Yeast Glucan Particles for Antibody-Free Separation of Viable Macrophages from Drosophila melanogaster.

Currently available methods for cell separation are generally based on fluorescent labeling using either endogenously expressed fluorescent markers or the binding of antibodies or antibody mimetics to surface antigenic epitopes. However, such modification of the target cells represents potential contamination by non-native proteins, which may affect further cell response and be outright undesirable in applications, such as cell expansion for diagnostic or therapeutic applications, including immunotherapy. We present a label- and antibody-free method for separating macrophages from living Drosophila based on their ability to preferentially phagocytose whole yeast glucan particles (GPs). Using a novel deswelling entrapment approach based on spray drying, we have successfully fabricated yeast glucan particles with the previously unachievable content of magnetic iron oxide nanoparticles while retaining their surface features responsible for phagocytosis. We demonstrate that magnetic yeast glucan particles enable macrophage separation at comparable yields to fluorescence-activated cell sorting without compromising their viability or affecting their normal function and gene expression. The use of magnetic yeast glucan particles is broadly applicable to situations where viable macrophages separated from living organisms are subsequently used for analyses, such as gene expression, metabolomics, proteomics, single-cell transcriptomics, or enzymatic activity analysis.

Isolation, structure characterization and in vitro immune-enhancing activity of a glucan from the peels of stem lettuce (Lactuca sativa).

BACKGROUND: Stem lettuce is a medicinal and edible plant. The peels, accounting for 300-400 g kg-1 raw stem lettuce and containing polysaccharides 200 g kg-1 , are discarded as industrial waste, causing environment pollution and resource waste. RESULTS: A polysaccharide named PPSL10-2 was obtained from the peels of stem lettuce after hot water extraction, and gradation with cascade ultrafiltration and purification using DEAE-Sepharose cellulose. The purity and molecular weight of PPSL10-2 is 96.10% and 2.2 × 104 Da respectively, as detected by high-performance gel permeation chromatography. PPSL10-2 was found to be an α-(1→4)-d-glucan that branched at O-6 with a terminal 1-linked α-d-Glcp as side chain, and devoid of helix conformation, which was characterized by monosaccharide composition analysis, Fourier-transform infrared spectroscopy, Congo red test, scanning electron microscopy, methylation analysis and NMR spectroscopy. Furthermore, PPSL10-2 exhibited potent immune-enhancing effect by improving proliferation and phagocytosis, promoting the secretion of nitric oxide and cytokines, as well as the expression of related genes in RAW264.7 macrophages. CONCLUSION: The findings of the present study suggest that peels as an agricultural by-product of stem lettuce are good sources of polysaccharides, which could be developed as immunopotentiator for improving human health. © 2023 Society of Chemical Industry.

Solid-State 13C Nuclear Magnetic Resonance Study of Soluble and Insoluble ß-Glucans Extracted from Candida lusitaniae.

ß-glucans are widely known for their biological activities. However, the choice of extraction method can significantly influence their structural characteristics, thereby potentially impacting their biological functions. In this paper, three fractions of ß-glucans were obtained from Candida lusitaniae yeast via alkali and hot-water extraction methods and were analyzed using solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR spectroscopy was used as a nondestructive technique that preserves the structure of the analyzed molecules. The results suggest that differences in the ß-glucan structure are affected by the choice of extraction method. The main difference occurred in the 82-92 ppm region with signal presence suggesting that ß-glucans have a linear structure when hot-water-extracted, which is absent in alkali-extracted fractions resulting in the acquisition of ß-glucans with an ordered, possibly helical structure. A hot-water extracted water-insoluble (HWN) fraction consists of linear ß-1,3-glucans with other signals indicating the presence of ß-1,6-linked side chains, chitin and small amounts of α-glucan impurities. For those that are alkali-extracted, alkali-insoluble (AN) and water-soluble (AWS) fractions are structurally similar and consist of an ordered ß-1,3-glucan structure with ß-1,6-linked side chains and a significant amount of α-glucan and chitin in both fractions.

Antitumor Activity of Carboxymethyl Pachymaran with Different Molecular Weights Based on Immunomodulatory and Gut Microbiota.

Carboxymethyl pachymaran (CMP) was treated via high-temperature and cellulase hydrolysis to obtain HTCMP, HTEC-24, and HTEC-48. The chemical structure and in vivo antitumor activities of the four types of CMPs were investigated. Compared with CMP (787.9 kDa), the molecular weights of HTCMP, HTEC-24, and HTEC-48 were decreased to 429.8, 129.9, and 68.6 kDa, respectively. The viscosities and particle sizes of the CMPs could also decrease with the decline in the molecular weights. All the CMPs showed antitumor abilities, but HTEC-24 exhibited the best activity. In the animal study, when curing the spleen and thymus, CMPs displayed immunomodulatory effects by increasing the secretion of IFN-γ and IL2 in mice. The CMPs also exerted an antitumor ability by regulating the gut microbiota in tumor-bearing mice. Our results established a foundation to develop an antitumor drug with CMP.

LAM2: An Unusual Laminaran Structure for a Novel Plant Elicitor Candidate.

Laminarans are of interest because they have been shown to induce various immune responses in animals and plants. These ß-D-glucans differ from each other by their branching rate, which is possibly responsible for their biological activities. In the present study, we characterized a laminaran fraction extracted from Laminaria hyperborea and named LAM2 using sugar composition and structural analyses (NMR). Then, we evaluated its activity as a potential plant elicitor in vitro on tomato seedlings using gene expression analysis and cell wall immunofluorescence labeling. Our study showed that LAM2 isolated from L. hyperborea is a succinylated laminaran which significantly enhanced the plant defense of tomato seedlings and induced cell wall modifications, suggesting a higher elicitor activity than the laminaran standard extracted from Laminaria digitata.

PURIFICATION, CHARACTERIZATION, AND IN VITRO ANTITUMOR ACTIVITY OF A NOVEL GLUCAN FROM PHOENIX DACTYLIFERA L. FRUITS.

In this study, ß- glucan was extracted by the hot water extraction method followed by ethanol precipitation and purified using ion and gel filtration chromatography, then evaluate the anticancer effects of ß- glucan that purified from Phoenix dactylifera on cancer cell line. Ahmed Nahi Glioblastoma Multiform (ANGM) cancer cell line was used in the in vitro study. Cell line exposure times were calculated after 24, 48, and 72 hours in a micro titration plate under absolutely sterile conditions. High molecular weight ß-glucans can be obtained using the hot water extraction method without having to use strong agents to change their structure, like alkalis or acids. Anti-cancer property of ß-glucan derived from Phoenix dactylifera fruits on cancer cell lines has been reported. In this work, the ANGM cell line was treated with different concentrations of ß-glucan (31.25, 62.5, 125, 250, 500 and 1000 µg/mL). and the inhibition of the cells was investigated using the MTT assay after 24, 48 and 72 hours. The result obtained showed time and concentration dependent cytotoxic effect, and the higher concentrations at 48 hrs of exposure gave significantly (p<0.05) higher cytotoxic effect.