Truncation of a novel C-terminal domain of a ß-glucanase improves its thermal stability and specific activity.

Enzymes that degrade ß-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-ß-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%-25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal ß-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-ß-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-ß-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

Antifungal characterizations of a novel endo-ß-1,6-glucanase from Flavobacterium sp. NAU1659.

ß-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of ß-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of ß-1,6-glucan by ß-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel ß-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-ß-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of ß-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: • ß-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • ß-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported ß-1, 6-glucanase derived from Flavobacterium.

Oat Beta-Glucan Dietary Intervention on Antioxidant Defense Parameters, Inflammatory Response and Angiotensin Signaling in the Testes of Rats with TNBS-Induced Colitis.

Male infertility represents a significant public health concern. There is a negative impact of inflammatory bowel diseases (IBDs) on the male reproductive system. The aim of this study was to investigate whether oat beta-glucan (OBG) with different molar mass can modulate parameters of antioxidant defense and inflammatory response in the testes of adult Sprague-Dawley rats with TNBS-induced colitis and whether the OBG intervention can modulate the inflammatory response in association with the RAS system. Results: higher testicular superoxide dismutase (SOD), glutathione reductase (GR) activities and glutathione (GSH) concentration, and lower testosterone (T) level and glutathione peroxidase (GPx) activity, were observed in rats with colitis than in healthy control ones. TNBS-induced colitis resulted in decreased the angiotensin 1-7 (ANG 1-7) level in the testes of rats fed with low-molar mass OBG compared to control animals. Conclusions: although colitis induced moderate pro-oxidant changes in the gonads, it seems plausible that dietary intervention with different fractions of oat beta-glucans mass may support the maintenance of reproductive homeostasis via the stimulation of the local antioxidant defense system.

ß-glucan: a potent adjuvant in immunotherapy for digestive tract tumors.

The immunotherapy for gastrointestinal tumors, as a significant research direction in the field of oncology treatment in recent years, has garnered extensive attention due to its potential therapeutic efficacy and promising clinical application prospects. Recent advances in immunotherapy notwithstanding, challenges persist, such as side effects, the complexity of the tumor immune microenvironment, variable patient responses, and drug resistance. Consequently, there is a pressing need to explore novel adjunctive therapeutic modalities. ß-glucan, an immunomodulatory agent, has exhibited promising anti-tumor efficacy in preclinical studies involving colorectal cancer, pancreatic cancer, and gastric cancer, while also mitigating the adverse reactions associated with chemotherapy and enhancing patients' quality of life. However, further clinical and fundamental research is warranted to comprehensively evaluate its therapeutic potential and underlying biological mechanisms. In the future, ß-glucan holds promise as an adjunctive treatment for gastrointestinal tumors, potentially bringing significant benefits to patients.

Enhancing Biomass and ß-Glucan Yield from Oyster Mushroom Pleurotus ostreatus (Agaricomycetes) Mycelia through Extract Valorization.

This study aimed to examine the impact of mushroom extract-based solid media on the ß-glucan content, growth rate, density, and biomass content of Pleurotus ostreatus (oyster mushroom) mycelia. Fresh, high-quality raw P. ostreatus were washed, sliced, and heated in a sealed pressure cooker at 90°C for 4 h in the drying cabinet. Following the heating process, centrifugation was carried out. Different concentrations of Pleurotus ostreatus extract were mixed with distilled water (0%, 25%, 50%, 75%, and 100%) and prepared for a sterile solid media. A malt extract-based medium was maintained as a control. This study focuses on the growth performance of P. ostreatus mycelium on its own mushroom extract-based culture medium which holds considerable economic and environmental significance. During the six-day observation period, the mycelium exhibited consistent growth across all tested media, maintaining a steady growth rate of 15 mm. The increased content of mushroom extract resulted from the enhanced density of the mycelia and biomass content. It can be inferred that when media containing less than 25% of mushroom extract dilution is used, ß-glucan can be formed in smaller amounts. Further research is needed to explore mushroom extract derived from different types of mushroom byproducts, which fail to meet commercial standards.

Size Matters: Influence of Particle Size on Antioxidant, ß-Glucan, and Anti-Inflammatory Potential in Pleurotus floridanus (Agaricomycetes).

Cellular damage resulting from elevated levels of free radicals can lead to persistent health issues. Pleurotus floridanus, an edible white oyster mushroom, is rich in ß-glucans with potent antioxidant and anti-inflammatory properties. In this research, we examined the ß-glucan content, total phenolic content, as well as antioxidant and anti-inflammatory potential of hot water extracts with varying particle sizes (< 75, 75-154, 154-300, and 300-600 µm) of both whole and sliced fruiting bodies of P. floridanus. The findings revealed that the в-glucan content increased as the particle size increased, although no significant differences were observed. Conversely, smaller particle sizes (< 75 µm) of whole and sliced fruiting bodies of P. floridanus exhibited higher phenolic content, 2,2-diphenyl-1-picryl-hy-drazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity, and reducing ability compared with larger particle size (> 75 µm). Of the four samples (AW2, AW3, AS1, and AS2) with the highest antioxidant activity selected for anti-inflammatory assays, all demonstrated the ability to reduce nitric oxide and tumor necrosis factor-alpha levels, but did not enhance interleukin-10 expression in lipopolysaccharide-stimulated RAW264.7 cells. Interestingly, particle size < 75 to 300 µm did not appear to influence the anti-inflammatory activity, because no significant differences were observed among the particle sizes. Therefore, a particle size < 300 µm in a P. floridanus hot water extract could serve as a valuable source of antioxidant and anti-inflammatory compounds to counteract the harmful effects of free radicals.

Alteration of ß-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence.

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as ß-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of ß-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface ß-glucan while low pH triggers an increase in ß-glucan. There is no inverse pattern between exposure levels of ß-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in ß-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in ß-glucan triggers an increase in the virulence of C. auris. This study demonstrates that ß-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Preparation and immunological activity evaluation of an intranasal protein subunit vaccine against ancestral and mutant SARS-CoV-2 with curdlan sulfate/O-linked quaternized chitosan nanoparticles as carrier and adjuvant.

Chitosan and its derivatives are ideal nasal vaccine adjuvant to deliver antigens to immune cells. Previously, we successfully used a chitosan derivative, O-(2-Hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (O-HTCC), and a ß-glucan derivative, curdlan sulfate (CS), to prepare a nanoparticle adjuvant CS/O-HTCC which could deliver ovalbumin to antigen presenting cells (APCs) through nasal inhalation. In this article, we used SARS-CoV-2 spike receptor binding domain (S-RBD) as the antigen and CS/O-HTCC nanoparticles as the adjuvant to develop a nasal mucosal protein subunit vaccine, CS/S-RBD/O-HTCC. The humoral immunity, cell-mediated immunity and mucosal immunity induced by vaccines were evaluated. The results showed that CS/S-RBD/O-HTCC could induce desirable immunization with single or bivalent antigen through nasal inoculation, giving one booster vaccination with mutated S-RBD (beta) could bring about a broad cross reaction with ancestral and different mutated S-RBD, and vaccination of the BALB/c mice with CS/S-RBD/O-HTCC containing S-RBD mix antigens (ancestral and omicron) could induce the production of binding and neutralizing antibodies against both of the two antigens. Our results indicate that CS/O-HTCC is a promising nasal mucosal adjuvant to prepare protein subunit vaccine for both primary and booster immunization, and the adjuvant is suitable for loading more than one antigen for preparing multivalent vaccines.

Improvement of mechanical, barrier properties, and water resistance of konjac glucomannan/curdlan film by zein addition and the coating for cherry tomato preservation.

The mechanical, barrier properties, and water resistance of packaging materials are crucial for the preservation of fruits and vegetables. In this study, zein was incorporated as a hydrophobic substance into the konjac glucomannan (KGM)/curdlan (KC) system. The KC/zein (KCZ) showed good compatibility with the zein aggregates uniformly distributed in the network formed by an entanglement of KGM and curdlan micelles based on hydrogen bonds. The presence of zein inhibited the extension of the KC entangled structure and enhanced the solid-like behavior. The high content of zein (>6 %) increased zein aggregation and negatively affected the structure and properties of KCZ. The zein addition significantly improved the water vapor permeability, tensile strength, and elongation at break. The hydrophobicity of the KCZ films was significantly enhanced, accompanied by the water contact angle increasing from 81° to 112°, and the moisture content, swelling, and soluble solid loss ratio decreasing apparently. The K56C40Z4 coating exhibited an excellent preservation effect to inhibit the respiration of cherry tomatoes, significantly reducing the water loss and firmness decline and maintaining the appearance, total solid, total acid, and ascorbic acid content. This work provided a strategy to fabricate hydrophobic packaging for the preservation of fruits and vegetables.

Effects of Nasal Solution Incorporating Resveratrol and Carboxymethyl-Β-Glucan in Preschool Non-Atopic Children with Wheezing.

Upper respiratory tract infections (URTI) account for more than 80% of wheezing episodes in children with a high incidence of hospitalization in preschool age. Most children with symptoms of wheezing during an URTI are usually non-atopic. As the majority of wheezing episodes resulting from URTI are attributed to viral triggers, several studies have suggested the potential anti-inflammatory and antiviral properties of resveratrol. This study aims to identify the effect of resveratrol for pediatric non-atopic patients with recurrent wheezing triggered by URTIs. We conducted a prospective single-blind study to assess the effectiveness of a short course of nasal solutions incorporating resveratrol and carboxymethyl-ß-glucan, administered for 7 days at the onset of URTIs, compared to standard nasal lavage with 0.9% saline solution. A total of 19 patients entered the active group, 20 patients were assigned to the placebo group. The comparison of overall wheezing days (p < 0.001), mean wheezing days per month (p < 0.01), and wheezing episodes per patient (p < 0.001) in the two groups showed a significant reduction in the group receiving resveratrol compared with the placebo group, with less hospital access (p < 0.001) and oral corticosteroid administration (p < 0.01). Our findings seem to suggest that, in non-atopic children with recurrent wheezing secondary to URTIs, nasal resveratrol could be effective to prevent or reduce the occurrence of wheezing, when started from the onset of upper airway symptoms.

Highly Soluble ß-Glucan Fiber Modulates Mechanisms of Blood Glucose Regulation and Intestinal Permeability.

ß-glucans found in cereal grains have been previously demonstrated to improve blood glucose control; however, current understanding points to their high viscosity as the primary mechanism of action. In this work, we present a novel, highly soluble, low-viscosity ß-glucan fiber (HS-BG fiber) and a preclinical dataset that demonstrates its impact on two mechanisms related to the prevention of hyperglycemia. Our results show that HS-BG inhibits the activity of two key proteins involved in glucose metabolism, the α-glucosidase enzyme and the SGLT1 transporter, thereby having the potential to slow starch digestion and subsequent glucose uptake. Furthermore, we demonstrate in a multi-donor fecal fermentation model that HS-BG is metabolized by several different members of the gut microbiome, producing high amounts of short-chain fatty acids (SCFAs), known agonists of GPR43 receptors in the gut related to GLP-1 secretion. The production of SCFAs was verified in the translational gut model, SHIME®. Moreover, HS-BG fiber fermentation produces compounds that restored permeability in disrupted epithelial cells, decreased inflammatory chemokines (CXCL10, MCP-1, and IL-8), and increased anti-inflammatory marker (IL-10), which could improve insulin resistance. Together, these data suggest that the novel HS-BG fiber is a promising new functional ingredient that can be used to modulate postprandial glycemic responses while the high solubility and low viscosity enable easy formulation in both beverage and solid food matrices.

Effects of Chronic Barley Consumption on Upper Respiratory Tract Symptoms in Japanese Healthy Adults: A Randomized, Parallel-Group, Controlled Trial.

ß-(1,3/1,4)-glucan is a major component of cereal grains, such as oats and barley. In this study, we investigated the effects of cooked waxy barley, which contains ß-(1,3/1,4)-glucan, on upper respiratory tract physical symptoms and mood status by performing a randomized, parallel-group, comparative trial. The primary outcome was assessed using the Wisconsin Upper Respiratory Symptom Survey-21 and Profile of Mood States second edition. Twenty-seven healthy Japanese adult participants were supplemented with 100 g of cooked waxy barley (containing 1.8 g of ß-glucan) or 100 g of cooked white rice daily for 8 weeks. Participants receiving cooked waxy barley reported a reduction in cumulative days of sneezing (p < 0.05) and feeling tired (p < 0.0001) compared with the control group. After the intervention period, there were significantly less severe nasal symptoms, such as runny nose, plugged nose, and sneezing (p < 0.05), and a significantly greater reduction of the Tension-Anguish score (p < 0.05) in the barley group than in the control group. This study suggests that supplementation of cooked waxy barley containing ß-(1,3/1,4)-glucan prevents or alleviates nasal upper respiratory tract symptoms and improves mood status. The findings of this study should be confirmed by double-blind trials with a larger number of participants.

Yeast ß-glucan-based nanoparticles loading methotrexate promotes osteogenesis of hDPSCs and periodontal bone regeneration under the inflammatory microenvironment.

The regeneration of absorbed alveolar bone and reconstruction of periodontal support tissue are huge challenges in the clinical treatment of periodontitis due to the limited regenerative capacity of alveolar bone. It is essential to regulate inflammatory reaction and periodontal cell differentiation. Based on the anti-inflammatory effect of baker's yeast ß-glucan (BYG) with biosafety by targeting macrophages, the BYG-based nanoparticles loading methotrexate (cBPM) were fabricated from polyethylene glycol-grafted BYG through chemical crosslinking for treatment of periodontitis. In our findings, cBPM promoted osteogenesis of human dental pulp stem cells (hDPSCs) under inflammatory microenvironment, characterized by the enhanced expression of osteogenesis-related Runx2 and activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/Erk) pathway in vitro. Animal experiments further demonstrate that cBPM effectively promoted periodontal bone regeneration and achieved in a better effect of recovery indicated by 19.2 % increase in tissue volume, 7.1 % decrease in trabecular separation, and a significant increase in percent bone volume and trabecular thickness, compared with the model group. Additionally, cBPM inhibited inflammation and repaired alveolar bone by transforming macrophage phenotype from inflammatory M1 to anti-inflammatory M2. This work provides an alternative strategy for the clinical treatment of periodontitis through BYG-based delivery nanoplatform of anti-inflammatory drugs.

Anti-Inflammatory Efficacy of Nanobody Specific to ß-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis.

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to ß-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

Microencapsulation of roasted mate tea extractives with lasiodiplodan (a (1 → 6)-ß-D-glucan) and maltodextrin as combined coating materials: A strategic tool to stabilize and protect the bioactive components.

Microencapsulation has emerged as a promising strategy to enhance the stability and protection of bioactive compounds. In this work, roasted mate tea was microencapsulated using 15 % maltodextrin and lasiodiplodan (0.5-1.25 %) as wall coating materials. The microcapsules were characterized for encapsulation efficiency, hygroscopicity, moisture, water activity, water solubility, dissolubility, scanning electron microscopy, FT-IR spectroscopy, thermal analysis, colorimetry, antioxidant activity, as well as quantification of phenolic compounds and caffeine. Microencapsulation yields ranged from 44.92 to 56.39 %, and encapsulation efficiency varied from 66.54 to 70.16 by increasing the lasiodiplodan concentration. FT-IR revealed phenolic acids, flavonoids, and polyphenolics. Minor color variations were observed among the samples. Thermal analysis demonstrated the microencapsulates exhibited good thermal stability with no degradation below 250 °C. Encapsulated samples showed high levels of bioactive compounds, suggesting that microencapsulation by spray-drying was a favorable process, where maltodextrin, a low-cost protective agent, when combined with the properties of lasiodiplodan, can be a good option for stabilizing mate extracts.

Swimming exercise alleviates pathological bone features in curdlan-injected SKG mice by inducing irisin expression.

This study assessed the therapeutic potential of swimming exercise in the curdlan-injected SKG mouse model and investigated the modulatory effects of irisin on inflammation. Curdlan-injected SKG were randomly assigned to either a home-cage group or a swimming group for 6 weeks. Changes in clinical arthritis scores and ankle thickness were measured weekly. Post-swimming program, mice were anesthetized for collection of vastus lateralis muscle and blood, which was followed by histological analysis, micro-CT imaging of the ankle joints, and the measurement of pro-inflammatory cytokines and irisin levels. Additionally, curdlan-injected SKG mice were intravenously injected with recombinant irisin protein and observed. Finally, serum levels of irisin in healthy control and ankylosing spondylitis (AS) patient groups were measured by ELISA. The swimming group of curdlan-injected SKG mice exhibited significant improvements in arthritis and enthesitis compared to the home-cage group. In particular, micro-CT and histological analyses revealed a notable reduction in pathological bone features in the swimming group compared to the home-cage group. Muscle endurance was also enhanced in the swimming group compared to the home-cage group, as determined by the wire-hanging test. Intriguingly, irisin levels not only were statistically increased in the swimming group but, also, TNF-α, IL-1ß, and IL-6 levels were decreased. Additionally, injection of irisin protein slightly attenuated both arthritis and enthesitis in curdlan-injected SKG mice. Meanwhile, irisin serum levels were declined in AS patients. Overall, we found that swimming exercise attenuated pathological bone features in an AS animal model, potentially mediated by increased irisin serum levels with associated anti-inflammatory effects.

Molecular mechanism underlying coencapsulating chrysophanol and hesperidin in octenylsuccinated ß-glucan aggregates for improving their corelease and bioaccessibility.

Chrysophanol and hesperidin are natural nutraceuticals that exhibit synergistic bioactivities, but their hydrophobicity limits their applications, and it is unclear whether coencapsulation can improve their solubility and release behaviors. The objective of this work was to coencapsulate chrysophanol and hesperidin by octenylsuccinated ß-glucan aggregates (OSßG-Agg) and to reveal how coencapsulation improves their release and bioaccessibility. Mechanisms underlying the hypothesis of beneficial effects in coloading, corelease and bioaccessibility were revealed. The solubilization of OSßG-Agg was due to hydrogen-bonding among ß-glucan moieties of OSßG and hydroxyl groups of chrysophanol and hesperidin and hydrophobic interactions among octenyl chains of OSßG and hydrophobic moieties of chrysophanol and hesperidin. Structural analyses confirmed the hypothesis that chrysophanol molecules were nearly embedded deeper into the interior of hydrophobic domains, and most of hesperidin molecules were incorporated into the exterior of the hydrophobic domains of OSßG-Agg due to the strength of these interactions, but they interacted in OSßG-Agg with a dense and compact structure rather than existing in isolation. The combined effects delayed their release and enhanced their bioaccessibility because of dynamic equilibrium between the favorable interactions and unfavorable structural erosion and relaxation of OSßG-Agg. Overall, OSßG-Agg is effective at codelivering hydrophobic phenolics for functional foods and pharmaceuticals.

Glycoside Phosphorylase Catalyzed Cellulose and ß-1,3-Glucan Synthesis Using Chromophoric Glycosyl Acceptors.

Glycoside phosphorylases are enzymes that are frequently used for polysaccharide synthesis. Some of these enzymes have broad substrate specificity, enabling the synthesis of reducing-end-functionalized glucan chains. Here, we explore the potential of glycoside phosphorylases in synthesizing chromophore-conjugated polysaccharides using commercially available chromophoric model compounds as glycosyl acceptors. Specifically, we report cellulose and ß-1,3-glucan synthesis using 2-nitrophenyl ß-d-glucopyranoside, 4-nitrophenyl ß-d-glucopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl ß-d-glucopyranoside with Clostridium thermocellum cellodextrin phosphorylase and Thermosipho africanus ß-1,3-glucan phosphorylase as catalysts. We demonstrate activity for both enzymes with all assayed chromophoric acceptors and report the crystallization-driven precipitation and detailed structural characterization of the synthesized polysaccharides, i.e., their molar mass distributions and various structural parameters, such as morphology, fibril diameter, lamellar thickness, and crystal form. Our results provide insights for the studies of chromophore-conjugated low molecular weight polysaccharides, glycoside phosphorylases, and the hierarchical assembly of crystalline cellulose and ß-1,3-glucan.

Effect of ß-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells.

ß-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of ß-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of ß-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. ß-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, ß-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, ß-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of ß-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a ß-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of ß-glucans on fish adaptive responses.