Biotech Application of Exopolysaccharides from Curvularia brachyspora: Optimization of Production, Structural Characterization, and Biological Activity.

C. brachyspora, a widespread dematiaceous fungus, was evaluated in this study to optimize the production of exopolysaccharides (CB-EPS). Optimization was performed using response surface methodology, and the best production yielded 75.05% of total sugar at pH 7.4, with 0.1% urea, after 197 h. The obtained CB-EPS showed typical signals of polysaccharides, which was confirmed by FT-IR and NMR. The HPSEC analysis indicated a polydisperse polymer, showing a non-uniform peak, with an average molar mass (Mw) of 24,470 g/mol. The major monosaccharide was glucose (63.9 Mol%), followed by mannose (19.7 Mol%), and galactose (16.4 Mol%). Methylation analysis encountered derivatives that indicated the presence of a ß-d-glucan and a highly branched glucogalactomannan. CB-EPS was tested on murine macrophages to verify its immunoactivity, and the treated cells were able to produce TNF-α, IL-6, and IL-10. However, the cells did not produce superoxide anions or nitric oxide nor stimulated phagocytosis. The results demonstrated an indirect antimicrobial activity of macrophages by stimulating cytokines, showing another biotech applicability for the exopolysaccharides produced by C. brachyspora.

MG-Pe: A Novel Galectin-3 Ligand with Antimelanoma Properties and Adjuvant Effects to Dacarbazine.

Melanoma is a highly metastatic and rapidly progressing cancer, a leading cause of mortality among skin cancers. The melanoma microenvironment, formed from the activity of malignant cells on the extracellular matrix and the recruitment of immune cells, plays an active role in the development of drug resistance and tumor recurrence, which are clinical challenges in cancer treatment. These tumoral metabolic processes are affected by proteins, including Galectin-3 (Gal-3), which is extensively involved in cancer development. Previously, we characterized a partially methylated mannogalactan (MG-Pe) with antimelanoma activities. In vivo models of melanoma were used to observe MG-Pe effects in survival, spontaneous, and experimental metastases and in tissue oxidative stress. Analytical assays for the molecular interaction of MG-Pe and Gal-3 were performed using a quartz crystal microbalance, atomic force microscopy, and contact angle tensiometer. MG-Pe exhibits an additive effect when administered together with the chemotherapeutic agent dacarbazine, leading to increased survival of treated mice, metastases reduction, and the modulation of oxidative stress. MG-Pe binds to galectin-3. Furthermore, MG-Pe antitumor effects were substantially reduced in Gal-3/KO mice. Our results showed that the novel Gal-3 ligand, MG-Pe, has both antitumor and antimetastatic effects, alone or in combination with chemotherapy.

Grape seed pomace as a valuable source of antioxidant fibers.

BACKGROUND: Grape seed pomace is a valuable by-product from winery/oil extraction industries and a potential source of bioactive compounds. Hot water extraction is the most widely used technology for polysaccharide recovery from plant and seed sources. To obtain an antioxidant fiber-rich extract (AFE) from defatted grape seed pomace, seed:water ratio (1:10-1:20) and extraction temperature (70-90 °C) effects on extraction yield, total sugars, total phenolic compounds and condensed tannins were investigated. RESULTS: The best extraction results were achieved at the highest seed:water ratio and temperature. At these conditions, the extraction yield and total sugar content of the freeze-dried AFE were 100.1 g kg-1 and 725.3 g kg-1 , respectively. The AFE presented high total phenolic content (16.2 g GAE kg-1 ), condensed tannins (515.1 mg CAT kg-1 ), soluble (219.1 g kg-1 ) and insoluble fibers (132.4 g kg-1 ), besides protein (171.1 g kg-1 ) and ash (152.6 g kg-1 ). The antioxidant capacity of the AFE was 382.7, 823.7 and 1439.4 µmol Trolox g-1 for DPPH, ABTS and ORAC methods. CONCLUSION: The AFE was shown to be not only an interesting source of fiber with antioxidant capacity, which could become a potential foodstuff, but also an ecofriendly and sustainable alternative to use grape seed pomace. © 2019 Society of Chemical Industry.

(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.

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.

All-polysaccharide, self-healing, pH-sensitive, in situ-forming hydrogel of carboxymethyl chitosan and aldehyde-functionalized hydroxyethyl cellulose.

In situ forming hydrogels are promising for biomedical applications, especially in drug delivery. The precursor solution can be injected at the target site, where it undergoes a sol-gel transition to afford a hydrogel. In this sense, the most significant characteristic of these hydrogels is fast gelation behavior after injection. This study describes an all-polysaccharide, rapidly in situ-forming hydrogel composed of carboxymethyl chitosan (CMCHT) and hydroxyethyl cellulose functionalized with aldehyde groups (HEC-Ald). The HEC-Ald was synthesized through acetal functionalization, followed by acid deprotection. This innovative approach avoids cleavage of pyran rings, as is inherent in the periodate oxidation approach, which is the most common method currently employed for adding aldehyde groups to polysaccharides. The resulting hydrogel exhibited fast stress relaxation, self-healing properties, and pH sensitivity, which allowed it to control the release of an encapsulated model drug in response to the medium pH. Based on the collected data, the HEC-Ald/CMCHT hydrogels show promise as pH-sensitive drug carriers.

Pressurized Liquid (PLE) Truffle Extracts Have Inhibitory Activity on Key Enzymes Related to Type 2 Diabetes (α-Glucosidase and α-Amylase).

An optimized PLE method was applied to several truffle species using three different solvent mixtures to obtain bioactive enriched fractions. The pressurized water extracts contained mainly (1 → 3),(1 → 6)-ß-D-glucans, chitins, and heteropolymers with galactose and mannose in their structures. The ethanol extracts included fatty acids and fungal sterols and others such as brassicasterol and stigmasterol, depending on the species. They also showed a different fatty acid lipid profile depending on the solvent utilized and species considered. Ethanol:water extracts showed interesting lipids and many phenolic compounds; however, no synergic extraction of compounds was noticed. Some of the truffle extracts were able to inhibit enzymes related to type 2 diabetes; pressurized water extracts mainly inhibited the α-amylase enzyme, while ethanolic extracts were more able to inhibit α-glucosidase. Tuber brumale var. moschatum and T. aestivum var. uncinatum extracts showed an IC50 of 29.22 mg/mL towards α-amylase and 7.93 mg/mL towards α-glucosidase. Thus, use of the PLE method allows o bioactive enriched fractions to be obtained from truffles with antidiabetic properties.

Investigation of the chemical structure and analgesic and anti-inflammatory properties of polysaccharides that constitute the dietary fibers of soursop (Annona muricata) fruit.

Soursop fruits are widely used in the folk medicine to treat a variety of health conditions. Once the chemical structure of dietary fibers from fruits is closely related to its biological functions in the human body, we aimed to explore structural features and biological activity of dietary fibers from soursop. Polysaccharides that constitute the soluble and insoluble fibers were extracted and further analyzed using monosaccharide composition, methylation, molecular weight determination and 13C NMR data. Soursop soluble fibers (SWa fraction) were characterized as having type II arabinogalactan and a highly methyl esterified homogalacturonan, while non-cellulosic insoluble fibers (SSKa fraction) were mainly composed by a pectic arabinan, a xylan-xyloglucan complex and a glucuronoxylan. The oral pre-treatment with SWa and SSKa promoted antinociception in mice writhing test, reducing the number of pain-like behaviors (in 84.2 % and 46.9 %, respectively, at 10 mg/kg) and peritoneal leucocyte migration (55.4 % and 59.1 %, at 10 mg/kg), effects possibly associated with the pectins present in fruit pulp extractions. SWa also significantly inhibited the plasmatic extravasation of Evans blue dye in 39.6 % at 10 mg/kg. This paper describes for the first time the structural features of soursop dietary fibers that may be of biological significance in future.

Analysis of Flavonoids from Eugenia uniflora Leaves and Its Protective Effect against Murine Sepsis.

Eugenia uniflora, referred to as Pitanga cherry shrub, is largely distributed in tropical and subtropical America. This plant is cultivated in many countries and it is suitable for the production of juice, frozen pulp, and tea. Besides, it can be used as treatment for inflammatory diseases. We reported that a flavonoid-rich fraction (HE-Bu) obtained from leaves decreased the lethality induced by cecal ligation and puncture (CLP), a clinically relevant model of sepsis. The oral administration of HE-Bu reduced the late mortality rate by 30%, prevented neutrophil accumulation in lungs, decreased TNF-α and IL-1ß serum levels, and markedly decreased iNOS and COX-2 protein expression by ileum cells. Chemical investigation showed myricetin and quercetin rhamnosides as the major components of this fraction. The results showed that HE-Bu protected mice from sepsis and indicated that this edible plant produces compounds that could be considered as potential adjuvants for sepsis treatment.

In vivo/in vitro studies of the effects of the type II arabinogalactan isolated from Maytenus ilicifolia Mart. ex Reissek on the gastrointestinal tract of rats.

Type II arabinogalactan (AG) is a polysaccharide found in Maytenus ilicifolia (Celastraceae), a plant reputed as gastroprotective. Oral and intraperitoneal administration of the AG protected rats from gastric ulcers induced by ethanol. No alteration of mechanisms related to acid gastric secretion and gastrointestinal motility were observed. In vitro, the AG showed a potent scavenging activity against the radical of DPPH (2,2-diphenyl-1-picrylhydrazyl) with an IC50 value of 9.3 microM. However, the mechanism of the gastroprotective action remains to be identified.

Antimelanoma effect of a fucoxylomannan isolated from Ganoderma lucidum fruiting bodies.

A fucoxylomannan (FXM) was isolated from the mushroom Ganoderma lucidum through alkaline extraction followed by dialysis, freeze-thawing, and fractionation by Fehling's solution. The main chain of FXM presented α-d-Manp-(1→4)-linked units, and some of them were branched at O-6 position by α-l-Fucp-(1→2)-ß-d-Xylp groups. Its Mw was 35.9 kDa. FXM was tested on melanoma B16-F10 cells and it showed cell viability and cell density reduction, as well as antiproliferative effect, through cell cycle arrest. Additionally, the anchorage-independent clonogenic capacity of such cells was significantly reduced by FXM, decreasing the number of cells by colony and the colonies area. No effect on viability neither in proliferation of non-tumoral Balb c/3T3 fibroblasts was observed. These results indicate that FXM is a promising anti-proliferative compound impairing pivotal tumorigenic mechanisms, eliciting this polysaccharide to be further explored as an antimelanoma drug.

A robust method to quantify low molecular weight contaminants in heparin: detection of tris(2-n-butoxyethyl) phosphate.

Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.

Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells.

BACKGROUND: Mushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells. METHODS: Mushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR. RESULTS: Semi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked ß-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of ß-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1ß and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract. CONCLUSIONS: The polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly ß-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.

Prebiotic potential of mushroom d-glucans: implications of physicochemical properties and structural features.

Mushroom d-glucans are recognized as dietary fibers and as biologically active natural polysaccharides, with the advantages of being quite inexpensive for production, tolerable, and having a range of possible structures and physicochemical properties. The prebiotic potential of mushroom d-glucans has been explored in recent years, but the relationship between their various structural features and activity is poorly understood. This review focuses on comprehensively evaluating the prebiotic potential of mushroom d-glucans in face of their structural variations. Overall, mushroom d-glucans provide a unique set of different structures and physicochemical properties with prebiotic potential, where linkage type and solubility degree seem to be associated with prebiotic activity outcomes. The understanding of the effects of distinct structures and physicochemical properties in mushroom d-glucans on the gut microbiota contributes to the design and selection of new prebiotics in a more predictable way.

Dietary Fiber Hierarchical Specificity: the Missing Link for Predictable and Strong Shifts in Gut Bacterial Communities.

Most dietary fibers used to shape the gut microbiota present different and unpredictable responses, presumably due to the diverse microbial communities of people. Recently, we proposed that fibers can be classified in a hierarchical way where fibers of high specificity (i.e., structurally complex and utilized by a narrow group of gut bacteria) could have more similar interindividual responses than those of low specificity (i.e., structurally simple and utilized by many gut bacteria). To test this hypothesis, we evaluated microbiota fermentation of fibers tentatively classified as low (fructooligosaccharides), low-to-intermediate (type 2 resistant starch), intermediate (pectin), and high (insoluble ß-1,3-glucan) specificity, utilizing fecal inoculum from distinct subjects, regarding interindividual similarity/dissimilarity in fiber responses. Individual shifts in target bacteria (as determined by linear discriminant analysis) confirmed that divergent fiber responses occur when utilizing both of the low-specificity dietary fibers, but fibers of intermediate and high specificity lead to more similar responses across subjects in support of targeted bacteria. The high-specificity insoluble ß-glucan promoted a large increase of the target bacteria (from 0.3 to 16.5% average for Anaerostipes sp. and 2.5 to 17.9% average for Bacteroides uniformis), which were associated with increases in ratios of related metabolites (butyrate and propionate, respectively) in every microbial community in which these bacteria were present. Also, high-specificity dietary fibers promoted more dramatic changes in microbial community structure than low-specificity ones relative to the initial microbial communities. IMPORTANCE In the face of interindividual variability and complexity of gut microbial communities, prediction of outcomes from a given fiber utilized by many microbes would require a sophisticated comprehension of all competitive interactions that occur in the gut. Results presented here suggest that high-specificity fibers potentially circumvent the competitive scope in the gut for fiber utilization, providing a promising path to targeted and predictable microbial shifts in different individuals. These findings are the first to indicate that fiber specificity is related to similarity and intensity of response in distinct human gut microbiota communities.

Microwave treatment enhances human gut microbiota fermentability of isolated insoluble dietary fibers.

Most insoluble dietary fibers are known to be relatively poorly fermented by the human gut microbiota. Here, the potential of microwave (MW) treatment to enhance the susceptibility of insoluble fruit polysaccharides to fermentation by the human gut microbiota was evaluated. Insoluble fruits dietary fibers before (xylan A, xylan T, and arabinan) and after MW (xylan A-MW, xylan T-MW, and arabinan-MW) treatment were fermented using an in vitro fermentation model. Gas production, shifts in pH, and short chain fatty acids (SCFAs) production showed an increase in fermentability of all tested dietary fibers, with an average 4-fold increase in SCFAs production after microwaving with total SCFAs ranging from 17.1 mM in the arabinan-MW to 40.4 mM in the xylan T-MW. While arabinan-MW and xylan T-MW promoted all three SCFAs proportionally (acetate:propionate:butyrate), xylan A-MW led to a marked and slow increase in butyrate reaching 28.1% of total SCFAs at 24 h. Rearrangements in three-dimensional structure that potentially facilitate bacterial accessibility to the dietary fiber were observed by scanning electron microscopy in xylan A-MW, forming coin-like particles with ~1.1 µm diameter. 16S rRNA gene sequencing indicated that microbiota shifts were related to both treatment (native versus MW) and dietary fiber type with many butyrogenic species being promoted by xylan A-MW. Overall, MW treatment enhanced insoluble dietary fiber fermentability promoting increased SCFAs production and bacterial shifts which are related to health benefits.

Polysaccharides from Pleurotus eryngii: Selective extraction methodologies and their modulatory effects on THP-1 macrophages.

Polysaccharides from P. eryngii mushroom were selectively extracted using low-cost technologies (water at different conditions of temperature and pressure). Mannogalactan was the main polysaccharide in cold-water extracted fraction (CWEF), while a linear (1→6)-ß-d-glucan was the main polymer in hot-water extracted fraction (HWEF). Autoclave-extracted fraction (AEF) contained a mixture of at least four different α- and ß-glucans. The report of linear (1→6)-ß-glucan and linear (1→3)-ß-glucan is a new finding for P. eryngii fruiting bodies. The immunostimulatory properties of the fractions on THP-1 macrophages were studied. All fractions at 50, 250 and 500 µg/mL were not cytotoxic and produced different stimulus on NO, IL-1ß and IL-10 secretion by the cells. Thus, our results showed that it is possible to concentrate different P. eryngii polysaccharides in selected fractions using a simple and low-cost procedure. Since biological effects depends on the polysaccharide structure, this technique allows the obtainment of fractions with distinct immunomodulatory activities.

Mucilages of cacti from Brazilian biodiversity: Extraction, physicochemical and technological properties.

Brazilian cacti have the high potential to be used as a source of mucilages with different technological functions that can be widely explored. The physicochemical, structural and technological properties of mucilages extracted from seven cladodes of cacti native from the semi-arid region of the Brazil were investigated. The mucilages exhibited high yields (8.9 to 21.54%), high contents of carbohydrates (39.77 to 87.68%), proteins (4.27 to 14.76%), and minerals, specifically Ca (2.90 to 15.65%). All species evaluated presented a diverse monosaccharide composition, mainly constituted of galactose and arabinose, suggesting the presence of arabinogalactans as the main polysaccharides. Both showed peaks and bands characteristic of polysaccharides with semi-crystalline structure (14.50-38.60%), due to the high concentration of calcium. From the results, mucilages functional properties showed good water and oil retention capacities and a high emulsification capacity (100%) with stability above 83%. Foaming capacity was above 58% with stability greater than 95%. This study highlights the potential of using cacti as a source of functional mucilages for use in food applications.

Gold nanoparticles capped with polysaccharides extracted from pineapple gum: Evaluation of their hemocompatibility and electrochemical sensing properties.

In the present work, gold nanoparticles were synthesized through a green route by using, for the first time, polysaccharides extracted from pineapple gum (PG) as the reducing and capping agent. The obtained nanoparticles (AuNPs-PG) were characterized by UV-VIS, FTIR, TEM, FESEM, EDX, XRD, and zeta potential measurements, which confirmed that PG was effective to produce AuNPs with an average diameter of 10.3 ± 1.6 nm. The AuNPs-PG were employed as the modifier of glassy carbon paste electrodes (CPE/AuNPs-PG), which were applied as sensitive electrochemical sensors to the determination of the antihistamine drug promethazine hydrochloride (PMZ). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements showed that the AuNPs-PG could enhance the electronic transfer properties of the glassy carbon paste, which was due to their large surface area and high electrical conductivity. After optimization of the instrumental parameters of square wave voltammetry (SWV) through a Box-Behnken factorial design, a linear relationship between the anodic peak current and PMZ concentration was obtained in the range from 2.0 to 15.7 µmol L-1 in McIlvaine buffer solution pH 5.0. The detection and quantification limits were found to be equal to 1.33 and 4.44 µmol L-1, respectively. The developed sensors could successfully quantify PMZ in different commercial pharmaceutical formulations, with satisfactory levels of accuracy and precision. In addition to improving the analytical features of the electrodes, hemocompatibility assays carried out on erythrocytes and leukocytes showed that the AuNPs-PG do not exhibit toxic effects on the referred cells. This interesting behavior enables their use in biocompatible electrochemical sensing platforms as well as for future biomedical investigations.

An α-D-galactan and a ß-D-glucan from the mushroom Amanita muscaria: Structural characterization and antitumor activity against melanoma.

Polysaccharides α-D-galactan (GAL-Am) and ß-D-glucan (GLC-Am) were obtained from Amanita muscaria fruiting bodies. They were purified using different methodologies, such as Fehling precipitation (for both fractions), freeze-thawing process and ultrafiltration (for GLC-Am). Results showed that the GAL-Am has (1 â†’ 6)-linked Galp main chain branched at O-2 by terminal Galp units and has not been previously reported. Besides, GLC-Am has (1 â†’ 3)-linked Glcp in the main chain, substituted at O-6 by (1 â†’ 6)-linked ß-Glcp units. Both are water-soluble, with 9.0 × 103 g/moL and 1.3 × 105 g/moL, respectively. GAL-Am and GLC-Am presented a selective proliferation reduction against B16-F10 melanoma cell line, not affecting non tumoral BALB/3T3 fibroblast cell line. Furthermore, both fractions reduced clonogenic capacity of melanoma cell line over an extended period of time. These results were obtained without modulations in B16-F10 cell adhesion, reinforcing the biological activities towards cell proliferation impairment and eliciting these polysaccharides as promising compounds to further exploration of their antimelanoma properties.