Production, characterization, and biological activity of a chitin-like EPS produced by Mortierella alpina under submerged fermentation

The production of a chitin-like exopolysaccharide (EPS) was optimized through experimental design methods, evaluating the influence of urea, phosphate, and glucose. Under optimized conditions, up to 1.51 g/L was produced and its physicochemical characteristics were evaluated by chromatography, NMR, and FTIR spectroscopy, and rheological techniques.

Carbohydrate Polymers 247 (2020) 116716 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Production, characterization, and biological activity of a chitin-like EPS produced by Mortierella alpina under submerged fermentation T Luis Daniel Goyzueta M.a, Miguel D Nosedab, Sandro J.R Bonattoc, Rilton Alves de Freitasd, Júlio Cesar de Carvalhoa,*, Carlos Ricardo Soccola a Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, CEP 81531-990, Curitiba, Paraná, Brazil Department of Biochemistry and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba, Paraná, Brazil c Faculdades Pequeno Príncipe, Curitiba, PR, Brazil d BioPol, Chemistry Department, Federal University of Paraná, P.B 19032, Centro Politécnico, CEP 81531-980 Curitiba, PR, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Mortierella alpina Chemical characterization Exopolysaccharide Chitin Antitumoral The production of a chitin-like exopolysaccharide (EPS) was optimized through experimental design methods, evaluating the influence of urea, phosphate, and glucose Under optimized conditions, up to 1.51 g/L was produced and its physicochemical characteristics were evaluated by chromatography, NMR, and FTIR spectroscopy, and rheological techniques The results showed a homogeneous EPS (Mw 4.9 × 105 g mol−1) composed of chitin, linear polymer of β-(1→4)-linked N-acetyl-D-glucosamine residues The acetylation degree as determined by 13C CP-MAS NMR spectroscopy was over 90 % The EPS biological activities, such as antioxidant effect and antitumor properties, were evaluated To the best of our knowledge, this is the first study on the production of a new alternative of extracellular chitin-like polysaccharide with promising bioactive properties from the filamentous fungus M alpina Introduction The fungus Mortierella alpina is well-known as a producer of arachidonic acid, a polyunsaturated fatty acid commonly used by different industries, such as food, medicine, cosmetics, and others, due to its nutraceutical properties (Ratledge, 2013) However, there is a lack of research on other bioactive substances produced by this fungus, such as polysaccharides Previous studies were carried out to found and identify polysaccharides from species of Mortierella, such as the research of Ruiter, Van Bruggen-Van Der Lugt, Rombouts and Gams (1993), in which a polysaccharide of M isabellina was characterized composed by 4-linked β-D-glucuronic acid residues The use of polysaccharides as antioxidant agents has been considered as a promising component in the formulation of effective, nontoxic drugs (Carocho & Ferreira, 2013; Ye, Liu, Wang, Wang, & Zhang, 2012) These polysaccharides can act in boosting the cell's natural defenses or by scavenging the free radical species (Sun, Wang, Fang, Gao, & Tan, 2004) Currently, there are several studies about the involvement of reactive oxygen species (ROS) in aging (Finkel & Holbrook, 2000) cancer and neurodegenerative disorders (Emerit, Edeas, & Bricaire, 2004) Additionally, the antitumor potential of these could be used in non⁎ aggressive drugs formulation (Gutierrez, Gonzalez, & Ramirez, 2012), and as additives for conventional cancer treatments This study aimed to introduce an eco-friendly approach to produce a chitin-like exopolysaccharide (EPS) by Mortierella alpina and to elucidate its chemical structure, the rheological properties, antioxidant activity, and antitumoral effects against tumor cell strains Materials and methods 2.1 Microorganisms and chemicals The fungal strain M alpina CBS 528.72 was purchased from the Centraalbureau voor Schimmelcultures (CBS, Netherlands) The culture was maintained on potato dextrose agar (PDA; glucose 20 g L−1, potato extract g L−1, and agar 17 g L−1) slants at ± °C and subcultured every months The monosaccharide standards (D-glucosamine, D-glucose, D-mannose, L-fucose, D-fructose, D-galactose, and D-glucuronic acid), chloramphenicol, 2,2′-azinobis-3-etilbenzothiazoline-6-sulfonic acid, 3-(2Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (Ferrozine), Iron (II) chloride, Disodium ethylenediaminetetraacetate dihydrate (EDTA-Na2), and 3-(4,5-dhimethylthiazol-2- Corresponding author E-mail address: jccarvalho@ufpr.br (J.C de Carvalho) https://doi.org/10.1016/j.carbpol.2020.116716 Received 13 May 2020; Received in revised form 15 June 2020; Accepted July 2020 Available online 03 July 2020 0144-8617/ © 2020 Elsevier Ltd All rights reserved Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al instead kDa) After exhaustive dialysis against ultrapure water, the suspension containing the EPS was freeze-dried, giving rise to the M alpina EPS fraction EPS (5 mg) was submitted to deacetylation using 50 % NaOH (5 mL) at 100 ± °C for 30 After the deacetylation process, the product was rinsed several times with hot distilled water and dried at 80 °C (Wojtasz-Pająk & Szumilewicz, 2009) Table Variables and their coded levels, 24 full factorial experimental design Independent Variables X1: X2: X3: X4: −1 Glucose (g L ) Urea (g L−1) pH Phosphate (as KH2PO4) (mmol L−1) Coded levels −1 40.0 2.0 5.0 1.5 60.0 4.0 6.0 3.8 +1 80.0 6.0 7.0 6.1 2.4 Partial acid hydrolysis yr)-2,5-diphenyltetrazolium bromide (MTT), 2,2′-azinobis-3-etilbenzothiazoline-6-sulfonic acid (ABTS), and 6-hydroxy-2,5,7,8- 23 tetramethylchroman-2-carboxylic acid (Trolox) were purchased from SIGMA-Aldrich The EPS was submitted to partial hydrolysis with the aim to reduce its high viscosity and allowed NMR analysis Briefly, the EPS (30.0 mg) partial hydrolysis was carried out with diluted TFA (0.1 mol L−1, 100 °C, h) (Wang et al., 2013) The partially hydrolyzed polysaccharide was dialyzed (MW cut off – kDa) against ultrapure water until no carbohydrates were detected in the dialysis water by conductivity (model CD-850, Lutron Electronic Enterprise Co., Taipei, Taiwan) The eluted (EF) and retentate (RF) solutions were freeze-dried Then, the monosaccharide composition of both fractions was performed following the methodology mentioned in the following Section 2.2 Culture medium and optimization of EPS production The culture medium composition for EPS production was in (g L−1): KNO3 1.0, MgSO4⋅7H2O 0.3, and in (mg L−1): CaCl2⋅2H2O 0.62, FeCl3⋅6H2O 1.5, ZnSO4⋅7 H2O 1.0, CuSO4⋅5H2O 0.1, and MnCl2⋅4H2O 1.0 Concentrations of glucose, urea, and phosphate at diverse pH levels were tested to evaluate their effect on EPS production (Mahapatra & Banerjee, 2013) In this first part of the optimization process, a 24 full factorial experimental design was used (16 experimental runs, with central points added to measure the intrinsic error) Experiments were performed randomly, and the results were analyzed at 95 % confidence intervals using the Statistica 7.0 software (StatSoft, Tulsa, OK, USA) In Table 1, four independent variables, their concentrations at different coded levels are shown The batch fermentation tests were carried out in 500 mL Erlenmeyer flasks (100 mL working volume) at 25 °C for days at 120 RPM, inoculated with 10 % (v.v−1) of a mycelial suspension The biomass was separated through vacuum filtration to obtain the filtrate broth EPS determination requires extraction, dialysis, and gravimetry, which incurs high errors for small batches Thus, viscosity was used as a proxy for evaluation of EPS production: a relation between the viscosity (mPa.s) and EPS produced (g L−1) (Fig 2S) was used (R2 = 0.98): EPS (g L−1) = 0.3254 *(X ) − 0.0204 * V , where X = Viscosity of the filtrate (mPa.s) and V = The total volume of the filtrate broth The viscosity was measured using an Ostwald viscometer, applying the following equation: ηL = ( ηW tL ρL )/(ρW tW ) where ƞW = Absolute viscosity of water, tW = Water flow time, ρW = Density of water, ƞL = Absolute viscosity of liquid, tL = Liquid flow time, and ρL = Density of liquid The second part of the optimization process used a central-composite design (Table 2) to obtain a response surface in the optimal region, with variables at coded levels Two axial points were chosen −1.681 and 1.681 to make the design orthogonal The software Statistica 7.0 (StatSoft, Tulsa, OK, USA) was used to analyze the results at a 95 % confidence interval 2.5 General analyses The EPS cleanliness, related to suspended solids and traces, was evaluated by UV–vis spectroscopy, recorded using a SHIMADZU (VIS1601PC, Tokyo, Japan) spectrophotometer, by dissolving EPS in LiCl 0.28 mol L−1, in the range of 200 and 800 nm Fourier-transform infrared (FTIR) spectra were recorded using KBr pellets of the EPS on an MB-series spectrophotometer (Bomem-Hartmann & Braun, Quebec, Canada) from 400 to 4000 cm−1 (64 scans, cm−1 resolution) The total protein content was measured using the Bradford method (Bradford, 1976), and uronic acids were determined by spectrophotometry (Filisetti-Cozzi & Carpita, 1991) Carbohydrate remaining after dialysis processes were determined by anthrone‐sulphuric acid assay, and glucose was used as a standard (Morris, 1948), and by conductivity (model CD-850, Lutron Electronic Enterprise Co., Taipei, Taiwan) 2.6 Monosaccharide composition analysis For monosaccharide composition determination, EPS was submitted to total acid hydrolysis (1 mol L−1 TFA, 100 °C, h), reduced (NaBH4, 16 h, 25 °C), acetylated (acetic anhydride 0.5 mL and sodium acetate as the catalyst, h, 100 °C) and analyzed as their alditol acetates derivatives by GC–MS/MS QP2010 model coupled to a TQ8040 tandem mass spectrometer (Shimadzu Corporation, Kyoto, Japan) with a Combi Palm AOC-5000 autosampler, SH-Rtx-5 ms column (30 m x0.25 mm x0.25 μm) The chromatograph was programmed to run from 100 to 250 °C (8 °C min−1), using He 99.99 % (1.0 mL min−1 constant flow) as the carrier gas The alditol acetates were identified by their typical electron-impact fragmentation profiles and GC retention times DGlucose, D-mannose, D-arabinose, D-galactose, D-xylose, D-fucose, and Dglucosamine were treated as samples and used as standards 2.3 EPS recovery After the fermentation process, the biomass was removed by vacuum filtration, and the EPS was recovered following a modified method described by Lima et al (2008) (dialysis: MW cut-off 20 kDa 2.7 Homogeneity and MW determination The EPS (1.0 mg mL−1) was dissolved in 0.1 mol L−1 NaNO2 containing NaN3 (0.2 g L−1) at 25 °C and filtered using 0.22 μm cellulose acetate membranes The biopolymer analysis was performed using a Waters high-pressure size-exclusion chromatography (HPSEC) system coupled to a multi-angle laser light scattering detector (Wyatt Technology Dawn DSP, Santa Barbara, CA, USA) and a differential refractive index detector (Waters 2410, Milford, MA, USA) The products were separated isocratically at 0.6 mL min−1, using four Waters Ultrahydrogel columns (Milford, MA, USA) with exclusion limits of 7.106, 4.105, 8.104, and 5.103 g mol−1 placed in series For the Table Central-composite experimental design, Variables, and their coded levels Independent Variables Glucose (g L−1) Urea (g L−1) Phosphate (as KH2PO4) (mmol L−1) Coded levels −1.681 −1 +1 +1.681 26.36 2.63 1.95 40.00 4.00 6.11 60.00 6.00 12.22 80.00 8.00 18.32 93.64 9.36 22.48 Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al refractive index increment (∂n/ ∂c ) determination, fractions were dissolved in five concentrations (0.2–1.0 mg mL−1) using the same eluent and filtered through a 0.22 μm cellulose membrane before injection The data were collected and analyzed with the Wyatt Technology ASTRA program (Santa Barbara, CA, USA) Table Yields, chemical analyses, and monosaccharide composition of M alpina EPS and its products of partial acid hydrolysis Parameters 2.8 Nuclear magnetic resonance (NMR) spectroscopy Partial acid hydrolysis fractions Yield (%) Mw (g mol−1)c For solid-state NMR, 13C CP-MAS spectra were recorded on a Bruker AVANCE 400 spectrometer (100.63 MHz for 13C nuclei) at 20 °C, equipped with a mm multinuclear probe with magic angle spinning (MAS) The EPS sample was humidified for days in a closed vessel containing a water-saturated atmosphere to enhance the spectrum resolution (Paradossi & Lisi, 1996) For NMR analyses, the partially hydrolyzed fractions were dissolved in 99.99 % D2O under ultrasonic treatment at 20 % amplitude (12 W cm−3) for 10 in an ice-water bath (Wang, Cheung, Leung, & Wu, 2010) The concentrations used were 40 mg mL−1 for 13C and 15 mg mL−1 for 1H and 2D NMR analyses and were recorded at 30 °C using a Bruker Avance DRX400 spectrometer (Bruker, Billerica, MA, USA) Chemical shifts were expressed relative to acetone (internal standard) at 31.45 and 2.225 ppm for 13C and 1H nuclei, respectively Uronic acids (%)d Proteins (%)e Monosaccharide composition (mol%) D-Glucosamine D-Glucose EPS Eluted (EF) Retained (RF) 7.5a 4.9 × 105 0.5 5b nd 95b nd nd nd nd nd 99 99 99 f nd Not determined a % EPS related to biomass b % fraction related to EPS c Determined by HPSEC-MALLS-RID d Filisetti-Cozzi & Carpita (1991) e (Bradford (1976) f Determined by GC–MS (see chromatogram in Fig 1D) in a lapse of 1.5 h 2.9 Acetylation and deacetylation degree analysis 2.12 Cell lines and culture conditions The degree of acetylation (DA) of the EPS was determined by NMR spectroscopy DA was calculated dividing the intensity of the methyl group carbon by the average intensity of the carbons (obtained from the 13 C CP-MAS NMR spectrum), following the equation (Vårum, Anthonsen, Grasdalen, & Smidsrød, 1991): %DA = 100xICH3/[(IC1 + IC + IC + IC + IC5 + IC 6)/6)], where I represents the intensity of the corresponding particular resonance peak Additionally, FTIR was also used to calculate the deacetylation degree from the absorption bands at 1320 (acetylated amine or amide function) and 1420 cm−1 (reference band) (Brugnerotto et al., 2001), A1320 following the equation: DD% = 100 − ( A1420 − 0.3822) * 1/0.03133 Breast cancer cell lines (MCF7, MDA-MB 231, and MDA-MB 468) and the control (MCF 10A) were purchased from the cell bank of Rio de Janeiro – Brazil and cultivated in Dulbecco's Modified Eagle’s Medium F12 (DMEM) supplemented with 10 % fetal bovine serum (FBS), except MCF7 (20 % of FBS) The cultivation of the non-tumorigenic epithelial cell line MCF10A was supplemented with 10 μg mL−1 human insulin, 0.5 μg mL−1 hydrocortisone, 10 ng mL−1 EGF, 100 ng mL−1 cholera toxin and 5% of horse serum instead of FBS The colorectal adenocarcinoma cell line CACO-2 was cultured in DMEM medium supplemented with 20 % of FBS Adrenocortical carcinoma H295R cell line (purchased from the ATCC bank) and the non-tumoral VERO cell line from kidney (purchased from the cell bank of Rio de Janeiro – Brazil) were also cultivated in DMEM medium supplemented with 10 % of FBS All cultivations contained 10 U mL−1 of streptomycin and 20 U mL−1 of penicillin For the assays, the cells were collected in a logarithmic growth stage using 0.6 % trypsin, and viability was evaluated using the trypan blue exclusion test The concentration of cells used was × 106 cells/well, pipetted in a 96-wells-flat-bottomed plate The incubation process was carried out for 24 h, 37 °C, and % CO2 humidified incubator 2.10 Antioxidant activity The ABTS radical scavenging assay was performed as described by Lee, Oh, Cho and Ma (2015) Trolox was used as a positive control and water as a blank The EPS samples were dissolved in ultrapure water to final concentrations of 0.5–5.0 mg L−1 and performed in triplicate The absorbance was measured using a PowerWave XS Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, USA) at 734 nm after 1, 5, and 10 of reaction The percentage of ABTS radical scavenging was calculated as shown % ABTS radical s in the following equation: cavenging = ((A0 − A1 )/ A0 ) x 100 , where A0 = absorbance control and A1 = absorbance of the sample 2.13 Growth inhibition assay The evaluation of the effect of the EPS on cell viability was carried out in the Research Institute Pelé Pequeno Príncipe – Curitiba – Paraná Brazil (IPPP) The EPS was solubilized at suitable conditions in ultrapure water by ultrasonic treatment at 20 % amplitude (12 W cm−3) for 10 in an ice-water bath (Wang et al., 2010) The cytotoxic effect was evaluated using the MTT assay The cell suspension (100 μL) was added to each well and incubated for 24 h, 37 °C and % CO2 After, the culture was replaced by 180 μL of fresh culture medium, and 20 μL of the EPS solution at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg L−1 were added After 24 h of incubation, 20 μL of the MTT solution was added to each well to a final concentration of 0.5 mg mL−1 Then, after h of reaction at 37 °C and 5%, CO2 100 μL of DMSO was added to each well Absorbance readings were performed at 595 nm on a microplate reader The viability of the untreated cell line group was considered as 100 % All assays were performed in five 2.11 Rheological studies The EPS was solubilized in LiCl 0.28 mol L−1 and NaCl 0.154 mol L Then, dynamic mechanical rheological measurements were carried out by monitoring visco-elastic moduli changes in the chitin-like solutions using a Thermo Scientific Haake Rheostress (Karlsruhe, Germany) equipped with a cone and plate geometry sensor (40 mm diameter, cone 2°) The gap between the plates was mm The loss (G’’) and storage (G’) shear moduli were in a wide range of frequencies (Hz) The imposed stress was chosen within the linear response regime (σ =0.1 Pa) unless otherwise specified Measurements were performed at 10–60 °C with an increment of °C ± 1, and the sensor was covered with a layer of mineral oil to avoid evaporation of the solutions The depercolation of gels was evaluated by cooling down from 60 to 10 °C −1 Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al Fig Chromatographic and spectroscopic analyses of M alpina EPS A) UV–vis scan spectrum; B) HPSEC-MALLS-RID elution profile (eluted with 0.1 M NaNO2 at a flow rate of 0.6 mL min−1); C) Deacetylated EPS (Red) and native EPS (Black) FTIR spectra, and D) GCMS of EPS product of total hydrolysis vs D-glucosamine standard (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Results and discussion replicates The following equation was used to calculate the percentage of viability: % cell viability = (100 x A595a )/A595b , where A595a is the mean value of the treatment samples and A595b is the mean value of the blanks 3.1 Culture medium and optimization of EPS production Previous work regarding culture medium optimization was carried Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al Fig Solid-state (13C CP-MAS) NMR spectrum of EPS produced by Mortierella alpina Fig HSQC NMR spectrum of the deacetylated EPS under alkaline conditions Solvent: D2O Temperature: 30 °C Acetone was used as an internal standard 31.45 and 2.225 ppm out to establish the best conditions for suitable biomass growth of M alpina (Goyzueta Mamani, 2014) Using the previously determined culture medium as a base, a new formulation optimized for EPS production was developed A 24 experimental design was used with the independent variables, biomass, and viscosity, as a proxy for evaluation of the EPS production The factors considered were: glucose (g L−1) (as X1), urea (g L−1) (as X2), pH (as X3), and phosphate (mmol L−1) (as X4) In this first part, the first-order polynomial equations obtained from the statistical regression for biomass production and viscosity enhancement are as follows: (1) −1 Biomass (g L ) = 18.17 + 2.85*X1 + 1.8*X4 + 1.15*X1X4 (2) Eq indicates that the increment of urea and phosphate concentrations enhanced EPS production Glucose and pH did not show a positive influence on viscosity enhancement In Eq 2, glucose and phosphate have shown a positive effect on biomass production Thus, glucose, urea, and phosphate were selected for the subsequent optimization process The pH was maintained at its minimum level In the second part of the optimization process, a central-composite design was carried out (Fig 1S); the experimental results are shown in Table 1S The second-order polynomial equation from the statistical Viscosity (mPa.s) = 1.53 + 0.5*X2 + 0.23*X4 – 0.1*X1X2 + 0.2*X2X4 Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al Fig 2D HSQC NMR spectrum of RF (retentate fraction) obtained from EPS partial acid hydrolysis Acetone was used as an internal standard at 31.45 and 2.225 ppm Fig Frequency dependence of the storage and loss moduli of chitin-like EPS-based solution at concentrations of A) mg mL−1, B) 10 mg mL−1 and C) 15 mg mL−1 of chitin in LiCl and D) mg mL−1, E) 10 mg mL−1 and F) 15 mg mL−1 of chitin in NaCl over a wide range of temperature Storage modulus is represented in filled symbols and Loss modulus in open symbols factors for a maximum predicted production determined were: glucose 46.01 g L−1; urea 7.48 g L−1, and phosphate 11.81 mM L−1, giving viscosity of 4.71 mPa.s, equivalent to 1.51 g L−1 of EPS (at 95 % confidence) When reproducing this model, the maximum production of 1.46 ± 0.11 g L−1 was obtained regression for viscosity (mPa.s) was as follows: Viscosity (mPa.s) = −7.83−0.002*X1 +1.795*X2 −0.025*X42 +0.354*X4 +0.162*X1 −0.117*X22 (3) The analysis of the t-test (Table 1S) showed that all the quadratic factors influenced positively on the viscosity enhancement (p < 0.05), hence the EPS production Using this mathematical model, the optimal concentration of the Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al 2D NMR analysis was also performed on the partially deacetylated EPS (Fig 3) The HSQC spectrum showed only one anomeric correlation at 98.7/4.88 ppm and a characteristic cross-peak at 57.0/3.19 attributed to C1/H1 and C2/H2 of β-linked glucosamine units, respectively The other well-defined correlations at 71.2/3.92, 77.5/3.92, 76.0/3.75, 61.2/3.76,3.93 ppm were attributed to C3/H3-C6/H6,H6′ of the same units, respectively The high-field correlation at 21.5/2.09 ppm corresponds to CH3 of the residual acetyl group All these assignments agree with the structure of a partially deacetylated chitin-like biopolymer 3.2.3 EPS partial acid hydrolysis M alpina EPS was submitted to partial acid hydrolysis giving rise to a high molecular mass retentate fraction (RF, in MW cutoff kDa) that presented glucosamine as main monosaccharide constituent (Table 3) The HSQC NMR spectrum of RF showed the following correlations at 101.7/4.54, 56.1/3.70, 69.6/3.50, 81.1/3.80, 76.4./3.46, 61.9/ 3.89,3.75 ppm attributed to C1/H1-C6/H6,H6′, respectively of β-(1→ 4)-linked D-glucosamine units (Fig 4) The methyl correlation at 23.7/ 2.02 ppm confirmed the N-acetylation of RF Summarizing, the chemical and spectroscopic analysis shows that the fungus M alpina biosynthesize an exopolysaccharide with a chitinlike structure Fig % scavenging activity of EPS evaluated at 1, 5, and 10 of reaction time 3.2 EPS characterization 3.2.1 General analyses Physical-chemical characterization analyses of M alpina EPS and its partial hydrolysis products are shown in Table EPS monosaccharide analysis showed the presence of glucosamine as a major constituent (Table 3) The EPS solution in LiCl (0.28 mol L−1) was transparent, and no precipitation occurred after 10 centrifugation at 14,000 RPM In the ultraviolet region, no significant absorbance of proteins was observed at 260 nm (Fig 1A) No presence of detritus was found in the visible range The EPS elution profile, evaluated by HPSEC-MALLS-RID showed a unique and symmetric peak (Fig 1B), indicating a homogeneous molar mass (Mw) distribution The EPS Mw as determined by HPSEC-MALLS∂n RID analysis was 4.9 × 105 g mol−1 ( ∂c = 0.121 mL g−1) The FTIR spectrum of EPS is shown in Fig 1C, in which some characteristic bands were observed at 3489 cm−1 attributed to OH groups, typical in polysaccharides (Duarte, Ferreira, Marvão, & Rocha, 2002), 3307 cm−1 attributed to NH2 groups, 2899 cm−1 attributed to an aliphatic C–H stretching band, the main characteristic of chitins (C] O stretching) attributed to the vibration of the amide I band at 1682 cm1 (Rumengan et al., 2014), 1574 cm−1 attributed to the NeH deformation of amide II, 1438 cm−1 attributed to the CH3 group deformation (Schenzel & Fischer, 2001) The band showed at 1097 cm−1 was attributed to the CeOeC glycosidic linkage vibration (Puspawati & Simpen, 2010) After deacetylation, the absorption band assigned to amide II decreases, while the increase of the intensity of amide I band indicates the formation of NH2 groups The EPS showed a higher intensity band of amide I than the band of amide II, suggesting an efficient deacetylation (Al Sagheer, Al-Sughayer, Muslim, & Elsabee, 2009) 3.3 Rheological studies The evaluation of gel formation as a response to a temperature increment was carried out to understand the behavior of chitin-like EPS solutions made of different concentrations and solubilized in NaCl and LiCl The frequency dependence of the storage and loss shear moduli at different temperatures (10–60 °C) is shown in Fig Similar behavior was noted between the 0.154 NaCl and 0.28 mol L−1 LiCl solutions were used as the solvents When a concentration of mg mL−1 of EPS was prepared (Fig 5A,D), a predominant liquid-like behavior was observed, with loss modulus (G”) higher than the storage modulus (G’) A solid-like behavior, due to gelation, was observed at 10 and 15 mg L−1 when the temperature was increased from 10 °C to 60 °C, observing high variations of the storage module (G’) at low frequencies (Fig 5B, E, C, F) This phenomenon was possibly caused by the formation of a permanent network of chitin chains, the formation of high-density cross-links population with long life, or permanent (Al-Muntasheri, Hussein, Nasr-El-Din, & Amin, 2007) According to Wientjes, Duits, Jongschaap and Mellema (2000), these crosslinks might be formed by specific hydrophobic interactions between the chains, explaining the low solubility in polar solvents The depercolation of gels formed was also evaluated in this work to know whether a thermo-reversible effect could exist Gels were cooled down from 60 °C to 10 °C in a lapse of 1.5 h, but no depercolation effect (breakage of bonds) was observed (Fig 3S), suggesting that long periods might be needed to attain this effect This highly suggests that the kinetic of depercolation is slower than the kinetic of percolation 3.2.2 NMR analyses M alpina EPS forms highly viscous solutions, and for this reason, it was not possible to obtain a good quality NMR spectrum of this biomolecule, even at high temperatures The 13C CP-MAS NMR spectrum of the native EPS (Fig 2) showed signals that were attributed to β(1→4)-linked N-acetyl-D-glucosamine residues, as follows: anomeric carbon at 101.71 ppm, C2 (55.0 ppm)(characteristic of GlcNAc), C4 (83.4 ppm), C5 (75.9 ppm), C3 (70.7 ppm), and C6 (62.1 ppm) Additionally, the signal at 23.6 ppm corresponds to CH3 of the acetyl group, and the signal at 174.6 ppm was attributed to the carbonyl group (Heux, Brugnerotto, Desbrières, Versali, & Rinaudo, 2000; Kono, 2004; Saitô, Tabeta, & Hirano, 1981; Younes & Rinaudo, 2015) The methyl carbon signal of the acetyl group suggests a high acetylation degree of the polysaccharide, over 90 % of acetylation was calculated by the intensities of the 13C CP-MAS NMR signals EPS was submitted to an alkali deacetylation process (Chen, Wang, & Ou, 2004), with the aim to improve the polysaccharide solubility for further chemical analyses and potential applications Deacetylation of 70 % was reached, as estimated by the FTIR spectrum (Fig 1C) 3.4 ABTS radical scavenging In this study, the half-maximal inhibitory concentration (IC50) calculated in of the assay was 2.08 mg mL−1, in which a final scavenging activity of 85 % was reached compared to 56 % of activity after of the assay at 2.5 mg mL−1 The determined Trolox Equivalent Antioxidant Capacity (TEAC) was 989.0 μmol equivalent.g−1 of chitin-like EPS (247 mg of Trolox.g−1 of chitin-like EPS It was noted that an increment of the EPS concentration resulted in an increment of the scavenging activity in short times (Fig 6) This phenomenon can be explained by the interaction of free radicals with the hydroxyl or amine groups of the chitin, forming stable macromolecule radicals (Xie, Xu, & Liu, 2001) The chitin-like EPS from M alpina, showed a promising higher Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al Fig Cell viability (%) of the EPS produced by M alpina on cells: A) H25R, B) CACO-2, C) VERO, D) MDA MB 231, E) MDA MB 468, F) MCF07, and G) MCF10A determined by the MTT assay Fig H shows the IC50 concentrations for tumor cells Carbohydrate Polymers 247 (2020) 116716 L.D Goyzueta M., et al Resources, Writing - review & editing antioxidant effect when compared to other chitin sources, such as Insecta (IC50: 10.91 mg mL−1, 40 % of scavenging activity) (Kaya et al., 2015) or Crustacea (IC50: mg mL−1, 85 % of scavenging activity) (Vinsova & Vavrikova, 2011) Acknowledgments The authors thank the funding agencies CNPq andCAPES and the Pelé Pequeno Principe Research institution M.D.N., R.A.F., J.C.C., and C.R.S are Research Members of CNPq Acknowledgments to the Nuclear Magnetic Resonance Unit of the Chemistry and Biochemistry Departments (UFPR) L.D.G.M acknowledges a Ph.D scholarship from PROEX project 3.5 Antitumoral effect of EPS In the research on new biomolecules with active antitumoral effects, fungal polysaccharides have demonstrated potential, but there are few studies about extracellular chitin specifically (Lenardon, Munro, & Gow, 2010) The MTT assay showed a significant inhibitory effect on cellular proliferation in all the tumoral cell lines after 24 h (Fig 7) and IC50 values when compared to the untreated controls Significant tumoral cell growth inhibition of > 50 % was observed at 1.37 mg mL−1 for H295R, 2.1 mg mL−1 for CACO-2, 1.5 mg mL−1 for MDA MB 231, 1.4 mg mL−1 for MDA MB 468 and 1.3 mg mL−1 for MCF 07, which means that the increment of EPS concentrations resulted in dose-dependent cell proliferation inhibition No growth inhibition over 50 % was observed in control at higher concentrations of EPS The in vitro studies of the effect of chitin on healthy cells demonstrated that the charges are essential for the antitumoral activity (Karagozlu, Karadeniz, Kong, & Kim, 2012), highly charged chitin compounds/derivatives triggered apoptotic pathways (Rinaudo, 2006) Adrenocortical carcinoma is an uncommon type of cancer treated with mitotane, an aggressive drug with collateral effects in infants (Gundgurthi et al 2012) For this reason, the cell line H295R was intentionally evaluated due to the number of cases in southern Brazil (Rodriguez‐Galindo, Figueiredo, Zambetti, & Ribeiro, 2005) The chitin-like EPS showed high potential as a candidate for further studies to evaluate the precise effect on tumor cells, especially in breast and colon cancer cells, aggressive cancer types when diagnosed in an advanced phase Chemotherapeutic agents could be formulated using different exopolysaccharides as additives, such as the one tested in this study Appendix A Supplementary 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solid-state NMR spectroscopy, which allows a direct analysis of the biopolymer and its structural elucidation Evaluation of the EPS toxicity against non-tumoral cell lines, such as VERO and MCF10A, provided the potential safeness utilization as an adjuvant in chemotherapeutics and chemopreventive drugs to fight adrenocortical carcinoma, breast, and colorectal cancer The capacity of M alpina EPS to produce hydrogels suggests the potential use of this chitin-like biopolymer as a biomaterial due to the formation of permanent cross-linked networks CRediT authorship contribution statement Luis Daniel Goyzueta M.: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft Miguel D Noseda: Methodology, Formal analysis, Writing - review & editing Sandro J.R Bonatto: Methodology, Formal analysis, Writing - review & editing Rilton Alves de Freitas: Methodology, Formal analysis Júlio Cesar de Carvalho: Conceptualization, Methodology, Formal analysis, Supervision, 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partial... chitin produced by the fungus Mortierella alpina as an antioxidant agent with antitumor activity, and potential use as a biomaterial A new study of the chitin-like EPS production by M alpina was carried... chemical analyses, and monosaccharide composition of M alpina EPS and its products of partial acid hydrolysis Parameters 2.8 Nuclear magnetic resonance (NMR) spectroscopy Partial acid hydrolysis fractions