Oligosaccharide Fractions Research Articles

Does Fungal Chitosan Leave Noticeable Traces in Treated Wines?

Background (1): The use of fungal chitosan as an antiseptic in wine appears as a promising alternative to sulfur dioxide for the elimination of Brettanomyces bruxellensis sensitive strains. Nevertheless, its utilization raises the question, "how are the treated wines different from the untreated ones?" Methods (2): Chitosan treatment residues were sought in the oligosaccharide and polysaccharide fractions and among 224 low MW ions (<1800 g·mol-1) in several wines by using liquid chromatography (size exclusion HPLC or LC-MS) and GC-MS. Standard oenological parameters were also examined as well as possible sensory modifications by a panel of tasters composed of experts and non-experts. Results (3): None of these methods enabled the reproducible and reliable identification of a treated wine without comparing it to its untreated control. The fingerprints of treatment are not reliably detectable by the analytical methods used in this study. However, the treated wines seem permanently protected against the development of chitosan-sensitive strains of B. bruxellensis. Conclusions (4): If chitosan treatment modifies the wine, the associated changes were not identified by the liquid chromatography method mentioned above and they were not perceived by most people in our taster panel. However, the expected antimicrobial action of chitosan was observed on B. bruxellensis sensitive strains and persisted at least one year. Tolerant strains were less affected by these persistent effects.

Physicochemical properties and immune enhancement activity of oligosaccharide fraction purified from biomass of green macro-algal Ulva prolifera

Ulva prolifera (U. prolifera), as the dominant species of green macro-algal bloom, caused great damage to marine aquaculture and economy. This study was carried out to find its industrial utilization ways by determination the structure characteristics and the immune enhancement effect of oligosaccharide fractions produced from this alga. The result indicated that oligosaccharide fraction of F3 showed the best immune enhancement activity via RAW 264.7 macrophages and zebrafish assays. Chemical and structural analysis indicated that F3 with 11.14%sulfate content was composed of rhamnose, xylose, and glucose in ratio of 77.11:16.33:6.56. NMR spectroscopy showed that F3 contained (1 → 4)-α-l-rhamnopyranose-3-SO3 and α-l-rhamnopyranose-3-SO3-(1 → 4)-β-D-xylopyranose, with sulfate groups located at C-3 of rhamnopyranose. The results of immune effect in vitro demonstrated that F3 increased the production of nitric oxide (NO) and prostaglandin E2 (PGE2) with up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. F3 also promoted the secretion of cytokines (TNF-α, IL-1β and IL-6) and activated the expression of mitogen-activated protein kinases (MAPK) and NF-κB signaling pathways. In vivo assay, F3 boosted the embryo survival rate and heart rate, as well as promoted the generation of NO and reactive oxygen species (ROS) in a dose-dependent manner. The novel structure and excellent activity in this study suggested that F3 could be the potential material for use as a natural immuno-enhancing agent in the biomedical and functional food industries.

Structural elucidation of mulberry leaf oligosaccharide and its selective promotion of gut microbiota to alleviate type 2 diabetes mellitus

Two oligosaccharide fractions (MLO 2-1 and 2-2) were purified from enzymatic hydrolysate of mulberry leaf polysaccharide. The results of simulated digestion showed that MLO 2-2 was a digestible oligosaccharide, which could be degraded by human digestive juice; while MLO 2-1 possessed the non-digestible property in the upper gastrointestinal tract and performed the function by regulating the gut microbiota. Hence, MLO 2-1 was selected for the further analysis. The structure of MLO 2-1 was elucidated as follow: α-T-Glcp-(1→3)-β-Glcp-(1→5)-α-Araf-(1→5) -α-Araf-1→5)-α-Araf-(1→3)-α-(2-OAc)-Glcp-1. The in vitro fecal fermentation results showed that MLO 2-1 could modulate the composition of gut microbiota. Meanwhile, MLO 2-1 was effectively metabolized by fecal bacteria to produce lactate and short chain fatty acids, especially acetate and butyrate. The specific metabolic pathways of MLO 2-1 by gut microbiota were further illuminated. Gut microbiota analysis revealed that MLO 2-1 selectively promoted the growth of Ligilactobacillus murinus, a commensal bacterium presented a reduced level in T2DM mice. Animal experiments indicated that MLO 2-1 and L. murinus exhibited hypoglycemic activities. These results demonstrated that MLO 2-1 might alleviate T2DM by selectively accelerating the proliferation of L. murinus.

Pichia pastoris Mediated Digestion of Water-Soluble Polysaccharides from Cress Seed Mucilage Produces Potent Antidiabetic Oligosaccharides.

Diabetes mellitus is a heterogeneous metabolic disorder that poses significant health and economic challenges across the globe. Polysaccharides, found abundantly in edible plants, hold promise for managing diabetes by reducing blood glucose levels (BGL) and insulin resistance. However, most of these polysaccharides cannot be digested or absorbed directly by the human body. Here we report the production of antidiabetic oligosaccharides from cress seed mucilage polysaccharides using yeast fermentation. The water-soluble polysaccharides extracted from cress seed mucilage were precipitated using 75% ethanol and fermented with Pichia pastoris for different time intervals. The digested saccharides were fractionated through gel permeation chromatography using a Bio Gel P-10 column. Structural analysis of the oligosaccharide fractions revealed the presence of galacturonic acid, rhamnose, glucuronic acid, glucose and arabinose. Oligosaccharide fractions exhibited the potential to inhibit α-amylase and α-glucosidase enzymes in a dose-dependent manner in vitro. The fraction DF73 exhibited strong inhibitory activity against α-amylase with IC50 values of 38.2 ± 1.12 µg/mL, compared to the positive control, acarbose, having an IC50 value of 29.18 ± 1.76 µg/mL. Similarly, DF72 and DF73 showed the highest inhibition of α-glucosidase, with IC50 values of 9.26 ± 2.68 and 50.47 ± 5.18 µg/mL, respectively. In in vivo assays in streptozotocin (STZ)-induced diabetic mice, these oligosaccharides significantly reduced BGL and improved lipid profiles compared to the reference drug metformin. Histopathological observations of mouse livers indicated the cytoprotective effects of these sugars. Taken together, our results suggest that oligosaccharides produced through microbial digestion of polysaccharides extracted from cress seed mucilage have the potential to reduce blood glucose levels, possibly through inhibition of carbohydrate-digesting enzymes and regulation of the various signaling pathways.

Effect of the Oligosaccharid Fraction of Fusarium oxysporum f. sp. vasinfectum on the Activity of Phenolic Metabolism Enzymes in Cotton Leaves

The synthesis of secondary metabolites, notably phenolic compounds, is part of the plant's defense reactions. The enzymes of phenolic metabolism can constitute a parameter of measurement of the defense of the plants. The aim of this work is to evaluate the activity of phenolic metabolism enzymes and an antioxidant enzyme after treatment of cotton plants with a fungal elicitor. Cotton plants obtained from the cultivar Y764AG3 were treated with the oligosaccharide fraction (FOS) of Fusarium oxysporum f. sp. Vasinfectum (FOV). The extraction of the enzymes is carried out cold by grinding the leaves in extraction buffer in the presence of PVP (0.5 %) and 0.1 M sodium phosphate buffer. The supernatant obtained after centrifugation of the filtrate was purified with Dowex-2 in order to obtain the purified enzymatic fraction. This fraction was used for the determination of enzyme activity by reading the absorbances. The results showed that the activity of ammonia-lyase activity was higher in the treated plants than in the control. This reflects an increase in the biosynthesis of phenolic compounds in the treated plants. On the other hand, oxidoreductase activity was higher in control plants than in treated plants. This indicates a decrease in the degradation of phenolic compounds in the treated plants. This study revealed the existence of an active phenolic metabolism in the FOS-treated plants. Thus defence mechanisms are activated in plants treated with the oligosaccharide fraction of Fusarium oxysporum f. sp. Vasinfectum.

Unmasking Chemokine-Inducing Specificity in Oligosaccharide Biomaterial to Promote Hair Growth.

Hair loss affects over 50 million people worldwide with limited therapeutic options. Despite evidence highlighting the vital role of local immune cells in regulating the life cycle of hair follicles (HFs), accurate regulation of immunocytes to directly promote hair growth remains unachieved. Here, inspired by the physiological feedback in the skin immunity to suppress microbe-triggered inflammation, an oligosaccharide biomaterial with "unmasked" specific activity is developed to recruit regulatory T (Treg ) cells around HFs, leading to accelerated hair growth in mice. By processing the glucomannan polysaccharide via controllable enzymatic cleavage, a series of oligosaccharide fractions with more specific chemokine-inducing functions is obtained. Notably, a hexasaccharide-based fraction (OG6) stimulates macrophages to selectively express Treg -chemoattractant C-C Motif Chemokine Ligand 5 (CCL5) through a mannose receptor-mediated endocytosis and NOD1/2-dependent signaling, as evidenced by molecular docking, inhibition assays, and a Foxp3-reporter mouse model. Intradermal delivery of OG6 to the depilated mouse skin promotes Treg mobilization around HFs and stimulates de novo regeneration of robust hairs. This study demonstrates that unmasking precise immunomodulatory functions in oligosaccharides from their parental polysaccharide can potentially solve the long-lasting dilemma with polysaccharide biomaterials that are widely renowned for versatile activities yet high heterogeneity, opening new avenues to designing glycan-based therapeutic tools with improved specificity and safety.

Isolation of a novel pentasaccharide Ebaliose from immunostimulant fraction of buffalo milk oligosaccharide

Buffalo milk processed by method of Kobata and Ginsburg for isolation of its oligosaccharide contents showed significant stimulation of anti-body, delayed type hypersensitivity response to sheep red blood cells in BALB/c mice. This oligosaccharide mixture also stimulated non-specific immune response in terms of MMI. In continuation to our previous studies of oligosaccharide contents of buffalo milk another novel pentasaccharide Ebaliose was isolated from immunostimulant oligosaccharide fraction of buffalo milk. Ebaliose was procured in its purest form, by combining the different chromatographic techniques like Gel filtration, Silica Gel column chromatography, and HPLC. The structure of novel oligosaccharide was determined by 1H,13C, HSQC, TOCSY, COSY, HMBC, ESI-MS, chemical transformation, and chemical degradation. The stereoscopic structure of this novel pentasaccharide was established as under.

Obtaining the Enoxaparin Sodium Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the “Greenness” of the Purification Stage

The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage involves the reprecipitation of crude enoxaparin in methanol. Determining the ratio of solvents required for the reprecipitation is important for studying the correlation between the experimental conditions of the technological process and the structural characteristics of enoxaparin samples. In the study, the method of purification of enoxaparin sodium described in the patent was assessed, and the following variations of the MeOH:H2O solvent ratio were selected – 4:1; 2:1; 1:1. The obtained samples of enoxaparin sodium were analyzed according to the in-house specification developed on the basis of the pharmacopoeial monograph, as well as by non-pharmacopoeial methods, such as two-dimensional NMR spectroscopy (HSQC) and size exclusion chromatography (SEC) for detailed characterization of the molecule. Strategies of greening of the enoxaparin sodium purification stage by reducing the E-factor were also considered in the study. Considering the principles of “green” chemistry, the method of purification of crude enoxaparin sodium was optimized by the solvent regeneration. It was experimentally possible to demonstrate the effect of the solvent ratio at the stage of purification of crude enoxaparin on the composition, as well as on the number and distribution of oligosaccharide fractions in the molecule. Based on the results of the study, it can be concluded that the ratio of MeOH:H2O=1:1 allows obtaining samples that are closest to Clexane® and Lovenox® in terms of the molecular weight distribution profile and the composition profile. The E-factor was also reduced from 14 to 5.25 by solvent regeneration.

Isolation and structural elucidation of prebiotic oligosaccharides from Ziziphi Spinosae Semen

Six oligosaccharides were discovered and isolated for the first time from Ziziphi Spinosae Semen. On the basis of spectroscopic analysis, their structures were determined to be verbascose (1), verbascotetraose (2), stachyose (3), manninotriose (4), raffinose (5), and melibiose (6). The prebiotic effect of the oligosaccharide fraction was assayed by eight gut bacterial growth in vitro, revealing a significant increase in cell density, up to 4-fold, for Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus johnsonii. The impact of six oligosaccharides with different degrees of polymerization (DPs) and structures on the growth of Lactobacillus acidophilus was evaluated. As a result, stachyose and raffinose demonstrated superior support for bacterial growth compared to the other oligosaccharides. This study explored the structure-activity relationship of raffinose family oligosaccharides (RFOs) and showed that the more the monosaccharide type, the more supportive the gut bacteria growth when oligosaccharides have the same molecular weight.

Fractionation of functional oligosaccharides produced from sugarcane straw using serial nanofiltration membranes and their influence on prebiotic potential

Functional oligosaccharides are non-digestible by human gut enzymes and provide health benefits as fibers and prebiotics. The cello-oligosaccharides (COS) and xylooligosaccharides (XOS) are functional oligosaccharides obtained from xylan and cellulose, respectively, and are present in lignocellulosic material. The serial NF membranes process was performed to investigate the impact of the fractionation process on the prebiotic activity of oligosaccharides from xylan and cellulose. The NP030 (weight cut-off of 500–600 Da) and DK (weight cut-off of 150–300 Da) NF polymeric membranes were employed using defined operational conditions. The diafiltration (DF) was also investigated and it was determined that only a 1-time DF for NP030 was a more suitable strategy and improved the performance indices for short DP oligosaccharides. The short DP fractions obtained favored cell density for probiotic strains, which presented an increase on the optical density of up to 25 % after the fractionating process; enabling the use of short purified fractions in the food and pharmaceutical industry as a prebiotic ingredient.

Structural requirements of Holothuria floridana fucosylated chondroitin sulfate oligosaccharides in anti-SARS-CoV-2 and anticoagulant activities.

Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.

Oligosaccharides from Human Milk Block Binding and Activity of the Escherichia coli Heat-Stable Enterotoxin (STa) in T84 Intestinal Cells , ,

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.

Optimization of an Innovative Hydrothermal Processing on Prebiotic Properties of Eucheuma denticulatum, a Tropical Red Seaweed

Seaweed is a sustainable source of marine oligosaccharides that potentially could be used as a prebiotic ingredient for functional food development. The study aims to optimize the oligosaccharide preparation through thermal hydrolysis of an under-utilized red seaweed, Eucheuma denticulatum. Response surface methodology (RSM) applying Box–Behnken design (BBD) was used on three parameters including temperature (105–135 °C), hydrolysis time (15–35 min) and sulfuric acid concentration (0.05–0.2 M). Optimized fractions with good prebiotic activity were characterized using high-performance size-exclusion chromatography (HP-SEC) and Fourier transform infrared spectroscopy (FT-IR). Eucheuma denticulatum oligosaccharides fraction 1 (ED-F1) was shown to promote the growth of beneficial gut microbiota including Lactobacillus plantarum, L. casei, L. acidophilus, Bifidobacterium animalis and B. longum with the highest prebiotic activity score of 1.64 ± 0.17. The optimization studies showed that hydrolysis time was the most significant parameter for the oligosaccharides yield. Optimal hydrolysis conditions for ED-F1 were 120 °C, 21 min, 0.12 M H2SO4 with the highest yield achieved (11.15 g/100 g of dry weight). The molecular weight of ED-F1 was determined at 1025 Da while FT-IR analysis revealed the presence of sulfated oligosaccharides with similar characteristics of i-carrageenan. These findings signify the innovative method for the efficient production of seaweed derived prebiotic oligosaccharides, which could be a promising source of functional food ingredients for the development of health foods and beverages.

Characterization of a novel GH26 β-mannanase from Paenibacillus polymyxa and its application in the production of mannooligosaccharides

A novel glycoside hydrolase family 26 β-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35–328) and PpMan26A-Δ205 (aa 206–656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The Km and kcat values for konjac gum were 2.13 mg/mL and 416.66 s–1, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3–8. CBM35pp exerted strong binding activity toward mannans but without β-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 β-mannanases and expands the potential industrial application of PpMan26A.

Preparation and Structural Analysis of a Sialo-Oligosaccharide from Glycophorin in Carp Red Blood Cell (RBC) Membranes

Glycophorins (GPs) in the red blood cell (RBC) membranes of carp (Cyprinus carpio L.) exhibit bacteriostatic activity against various gram-negative and gram-positive bacteria. This antibacterial property is attributed to a sialo-oligosaccharide. For a structural analysis of this O-linked sialo-oligosaccharide, it is necessary to prepare and purify the oligosaccharide from carp GPs. To prepare sialo-oligosaccharides, RBC membrane proteins were solubilized, and anion exchange column chromatography was performed to obtain the GPs. The GP yield from carp RBC membranes was ca.100% as total sialic acid. Then, released oligosaccharides were obtained from GP preparation by β-elimination. Second, the resulting oligosaccharide fraction was separated into two sialo-oligosaccharide components (P-1 and P-2) using the same anion exchange column. The oligosaccharide preparations that contained high amounts of NaCl were desalted with an activated charcoal column combined with ammonium bicarbonate in acetonitrile as an eluent. Using NMR to analyse the structure of the antimicrobial P-1, we determined the alignment of each proton and C1 carbon. To determine the linkage sites, we performed GC-MS using the permethylated P-1. Lastly, we determined that the structure of P-1 was NeuGcα2→6 (Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol.

GIPS-Mix for Accurate Identification of Isomeric Components in Glycan Mixtures Using Intelligent Group-Opting Strategy.

Accurate identification of glycan structures is highly desirable as they are intimately linked to their different functions. However, glycan samples generally exist as mixtures with multiple isomeric structures, making assignment of individual glycan components very challenging, even with the aid of multistage mass spectrometry (MSn). Here, we present an approach, GIPS-mix, for assignment of isomeric glycans within a mixture using an intelligent group-opting strategy. Our approach enumerates all possible combinations (groupings) of candidate glycans and opts in the best-matched glycan group(s) based on the similarity between the simulated spectra of each glycan group and the acquired experimental spectra of the mixture. In the case that a single group could not be elected, a tie break is performed by additional MSn scanning using intelligently selected precursors. With 11 standard mixtures and 6 human milk oligosaccharide fractions, we demonstrate the application of GIPS-mix in assignment of individual glycans in mixtures with high accuracy and efficiency.

Safety of 6'-sialyllactose (6'-SL) sodium salt produced by derivative strains of Escherichia coli BL21 (DE3) as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panelon Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on 6'-sialyllactose (6'-SL) sodium salt as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is mainly composed of the human-identical milk oligosaccharide (HiMO) 6'-SL, but it also contains d-lactose, 6'-sialyllactulose, sialic acid, N-acetyl-d-glucosamine and a small fraction of other related oligosaccharides. The NF is produced by fermentation with two genetically modified strains of Escherichia coli BL21 (DE3), the production strain and the optional degradation strain. The information provided on the identity, manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF to a variety of foods, including infant formula and follow-on formula, food for special medical purposes and food supplements. The target population is the general population. In some scenarios at the maximum use levels, the estimated intakes per kg body weight were higher than the high average natural intake of 6'-SL from human milk. However, given the intrinsic nature of human milk oligosaccharides (HMOs), the wide range of intakes from human milk, and considering that infants are naturally exposed to similar amounts of these substances, the Panelconsiders that the consumption of the NF at the proposed conditions of use does not raise safety concerns. The intake of 6'-SL in breastfed infants on a body weight basis is also expected to be safe for other population groups. The intake of other carbohydrate-type compounds structurally related to 6'-SL is also considered of no safety concern. Food supplements are not intended to be used if other foods with added 6'-SL or human milk are consumed on the same day. The Panelconcludes that the NF is safe under the proposed conditions of use.

Antimicrobial and prebiotic activity of mannoproteins isolated from conventional and nonconventional yeast species—the study on selected microorganisms

Yeast mannoproteins are proposed as a paraprobiotics with antimicrobial and prebiotic properties. They can be used as biopreservatives in food and in diseases therapies. The knowledge about the specificity and/or capability of their influence on the growth of different microorganism is limited. The study determined the effect of mannoprotein preparations of Saccharomyces cerevisiae (S. cerevisiae) ATCC 7090 and nonconventional yeast origin [Metschnikowia reukaufii (M. reukaufii) WLP 4650 and Wickerhamomyces anomalus (W. anomalus) CCY 38-1-13] on the growth of selected bacteria of the genera: Lactobacilllus, Limosilatobacillus, Limosilatobacillus, Bifidobacterium, Staphylococcus, Enterococcus, Pseudomonas, Escherichia, Proteus and Salmonella. The degree of stimulation or growth inhibition of tested bacteria depended on the type and dose of the mannoprotein and the bacterial strain. The addition of the tested preparations in the entire range of applied concentrations had a positive effect especially on the growth of Lactobacillus arabinosus ATCC 8014 and Bifidobacterium animalis subsp. lactis B12. Mannoproteins isolated from S. cerevisiae limited the growth of the Escherichia coli (E. coli) ATCC 25922, Pseudomonas aureoginosa (P. aureoginosa) ATCC 27853, Proteus mirabilis ATCC 35659 and Salmonella Enteritidis ATCC 13076 to the greatest extent, while preparations of M. reukaufii and W. anomalus origin most effectively limited the growth of Staphylococcus aureus strains, E. coli and P. aureoginosa. The growth of Enterococcus faecalis was stimulated by the presence of all studied preparations in most of the concentrations used. Further research will determine how the purification process of studied mannoproteins or oligosaccharide fractions, its structure and composition influence on the growth of selected bacteria and what is the mechanism of its activity.

Anti-SARS-CoV-2 and anticoagulant properties of Pentacta pygmaea fucosylated chondroitin sulfate depend on high molecular weight structures.

Fucosylated chondroitin sulfate (FucCS) is a unique marine glycosaminoglycan that exhibits diverse biological functions, including antiviral and anticoagulant activity. In previous work, the FucCS derived from Pentacta pygmaea (PpFucCS) showed moderate anticoagulant effect but high inhibitory activity against the Wuhan strain of severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this study, we perform free-radical depolymerization of PpFucCS by the copper-based Fenton method to generate low molecular weight (MW) oligosaccharides. PpFucCS oligosaccharides were structurally analyzed by 1H nuclear magnetic resonance spectroscopy and were used to conduct structure-activity relationship studies regarding their effects against SARS-CoV-2 and clotting. Anticoagulant properties were measured by activated partial thromboplastin time, protease (factors Xa and IIa) inhibition by serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]), and competitive surface plasmon resonance (SPR) assay using AT, HCII, and IIa. Anti-SARS-CoV-2 properties were measured by the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 Delta variant spike (S)-protein and competitive SPR assays using multiple S-proteins (Wuhan, N501Y [Alpha], K417T/E484K/N501Y [Gamma], L542R [Delta], and Omicron [BA.2 subvariant]). Cytotoxicity of native PpFucCS and oligosaccharides was also assessed. The PpFucCS-derived oligosaccharide fraction of the highest MW showed great anti-SARS-CoV-2 Delta activity and reduced anticoagulant properties. Results have indicated no cytotoxicity and MW dependency on both anti-SARS-CoV-2 and anticoagulant effects of PpFucCS, as both actions were reduced accordingly to the MW decrease of PpFucCS. Our results demonstrate that the high-MW structures of PpFucCS is a key structural element to achieve the maximal anti-SARS-CoV-2 and anticoagulant effects.

Deaminative-cleaved S. monotuberculatus fucosylated glycosaminoglycan: Structural elucidation and anticoagulant activity

Stichopus monotuberculatus is a tropical sea cucumber species and used as a folk medicine and tonic food. In this study, a fucosylated glycosaminoglycan (SmFG), the depolymerized SmFG (dSmFG) and its oligosaccharide fractions were prepared. The SmFG and its depolymerized products were comprised of a chondroitin-sulfate-E backbone, and various sulfated fucose side chains, including an unusual disaccharide side chain connected to the C-3 position of D-glucuronic acid (GlcA) or GlcA-ol. A peeling reaction occurred during the deaminative depolymerization process. The dSmFG and its fractions showed strong anticoagulant activity by selectively inhibiting intrinsic tenase complex, and had no anti-factor IIa, Xa and VIIa activity. The anticoagulant activity reduced with the decrease of molecular weight, and the unusual branch and novel reducing end may enhance the anticoagulant activity. These findings can provide significant information for development and utilization of depolymerized products from SmFG in food and pharmaceutical industries.