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.
Trang 1Contents lists available atScienceDirect
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
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
b 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
A R T I C L E I N F O
Keywords:
Mortierella alpina
Chemical characterization
Exopolysaccharide
Chitin
Antitumoral
A B S T R A C T 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 spectro-scopy, and rheological techniques The results showed a homogeneous EPS (Mw 4.9 × 105g mol−1) composed
of chitin, linear polymer ofβ-(1→4)-linked N-acetyl-D-glucosamine residues The acetylation degree as de-termined by13C 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 thefilamentous fungus M alpina
1 Introduction
The fungus Mortierella alpina is well-known as a producer of
ara-chidonic 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-linkedβ-D-glucuronic acid residues
The use of polysaccharides as antioxidant agents has been
con-sidered as a promising component in the formulation of effective,
non-toxic drugs (Carocho & Ferreira, 2013;Ye, Liu, Wang, Wang, & Zhang,
2012) These polysaccharides can act in boosting the cell's natural
de-fenses or by scavenging the free radical species (Sun, Wang, Fang, Gao,
& Tan, 2004) Currently, there are several studies about the
involve-ment 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 eluci-date its chemical structure, the rheological properties, antioxidant ac-tivity, and antitumoral effects against tumor cell strains
2 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 4 g L−1, and agar 17 g L−1) slants at 5 ± 1 °C and subcultured every 2 months
The monosaccharide standards (D-glucosamine, D-glucose, D-man-nose, L-fucose, D-fructose, D-galactose, and D-glucuronic acid), chlor-amphenicol, 2,2′-azinobis-3-etilbenzothiazoline-6-sulfonic acid, 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (Ferrozine), Iron (II) chloride, Disodium ethylenediami-netetraacetate dihydrate (EDTA-Na2), and
3-(4,5-dhimethylthiazol-2-https://doi.org/10.1016/j.carbpol.2020.116716
Received 13 May 2020; Received in revised form 15 June 2020; Accepted 1 July 2020
⁎Corresponding author
E-mail address:jccarvalho@ufpr.br(J.C de Carvalho)
Available online 03 July 2020
0144-8617/ © 2020 Elsevier Ltd All rights reserved
T
Trang 2yr)-2,5-diphenyltetrazolium bromide (MTT),
2,2′-azinobis-3-etilben-zothiazoline-6-sulfonic acid (ABTS), and 6-hydroxy-2,5,7,8- 23
tetra-methylchroman-2-carboxylic acid (Trolox) were purchased from
SIGMA-Aldrich
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 thisfirst part of the optimization process, a 24full
factorial experimental design was used (16 experimental runs, with 3
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 5 days at 120 RPM,
in-oculated with 10 % (v.v−1) of a mycelial suspension The biomass was
separated through vacuumfiltration 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: η L= (η W t ρ L L)/(ρ W t W)where ƞW =
Absolute viscosity of water, tW= Waterflow time, ρW= Density of
water,ƞL= Absolute viscosity of liquid, tL= Liquidflow time, and ρL
= Density of liquid
The second part of the optimization process used a
central-compo-site design (Table 2) to obtain a response surface in the optimal region,
with 3 variables at 5 coded levels Two axial points were chosen
−1.681 and 1.681 to make the design orthogonal The software
Sta-tistica 7.0 (StatSoft, Tulsa, OK, USA) was used to analyze the results at a
95 % confidence interval
2.3 EPS recovery
After the fermentation process, the biomass was removed by
va-cuum filtration, and the EPS was recovered following a modified
method described byLima et al (2008)(dialysis: MW cut-off 20 kDa
instead 8 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 ± 1 °C for 30 min After the deacetylation process, the product was rinsed several times with hot distilled water and dried at 80 °C (Wojtasz-Pająk & Szumilewicz, 2009)
2.4 Partial acid hydrolysis
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, 1 h) (Wang et al., 2013) The partially hydrolyzed polysaccharide was dialyzed (MW cut off – 1 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
2.5 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, 4 cm−1resolution) The total protein content was measured using the Bradford method (Bradford, 1976), and uronic acids were determined by spectro-photometry (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−1TFA, 100 °C, 4 h), reduced (NaBH4,
16 h, 25 °C), acetylated (acetic anhydride 0.5 mL and sodium acetate as the catalyst, 1 h, 100 °C) and analyzed as their alditol acetates deri-vatives 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 x0.25μm) The chromatograph was programmed to run from 100 to
250 °C (8 °C min−1), using He 99.99 % (1.0 mL min−1constantflow) as the carrier gas The alditol acetates were identified by their typical electron-impact fragmentation profiles and GC retention times D-Glucose,D-mannose, D-arabinose, D-galactose, D-xylose, D-fucose, and D-glucosamine were treated as samples and used as standards
2.7 Homogeneity and MW determination
The EPS (1.0 mg mL−1) was dissolved in 0.1 mol L−1NaNO2 con-taining NaN3(0.2 g L−1) at 25 °C andfiltered 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 re-fractive 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 1
Variables and their coded levels, 24full factorial experimental design
Independent Variables Coded levels
X 1 : Glucose (g L−1) 40.0 60.0 80.0
X 2 : Urea (g L−1) 2.0 4.0 6.0
X 4 : Phosphate (as KH 2 PO 4 ) (mmol L−1) 1.5 3.8 6.1
Table 2
Central-composite experimental design, Variables, and their coded levels
Independent Variables Coded levels
−1.681 −1 0 +1 +1.681 Glucose (g L−1) 26.36 40.00 60.00 80.00 93.64
Urea (g L−1) 2.63 4.00 6.00 8.00 9.36
Phosphate (as KH 2 PO 4 ) (mmol L−1) 1.95 6.11 12.22 18.32 22.48
Trang 3refractive index increment (∂n/ c) determination, fractions were dis-∂
solved infive concentrations (0.2–1.0 mg mL−1) using the same eluent
andfiltered 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)
2.8 Nuclear magnetic resonance (NMR) spectroscopy
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 4 mm multinuclear probe with magic angle spinning
(MAS) The EPS sample was humidified for 3 days in a closed vessel
containing a water-saturated atmosphere to enhance the spectrum
re-solution (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 min in an ice-water bath (Wang, Cheung, Leung, & Wu,
2010) The concentrations used were 40 mg mL−1for13C and 15 mg
mL−1for1H 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 for13C and1H nuclei, respectively
2.9 Acetylation and deacetylation degree analysis
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
13C CP-MAS NMR spectrum), following the equation (Vårum,
% 100 CH3/[(C1 C2 C3 C4 C5 C6)/6)], where I
re-presents the intensity of the corresponding particular resonance peak
Additionally, FTIR was also used to calculate the deacetylation
de-gree from the absorption bands at 1320 (acetylated amine or amide
function) and 1420 cm−1(reference band) (Brugnerotto et al., 2001),
following the equation:DD%=100−(A
A
1320
1420 −0.3822) * 1/0.03133 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−1and performed in triplicate The
absorbance was measured using a PowerWave XS Microplate
Spectro-photometer (BioTek Instruments, Inc., Winooski, USA) at 734 nm after
1, 5, and 10 min of reaction
The percentage of ABTS radical scavenging was calculated as shown
cavenging ((A0 A1)/A0) 100x , where A0= absorbance control and
A1= absorbance of the sample
2.11 Rheological studies
The EPS was solubilized in LiCl 0.28 mol L−1and NaCl 0.154 mol
L−1 Then, dynamic mechanical rheological measurements were carried
out by monitoring visco-elastic moduli changes in the chitin-like
solu-tions using a Thermo Scientific Haake Rheostress 1 (Karlsruhe,
Germany) equipped with a cone and plate geometry sensor (40 mm
diameter, cone 2°) The gap between the plates was 1 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 5 °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
in a lapse of 1.5 h
2.12 Cell lines and culture conditions
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 10μg mL−1human insulin, 0.5μg mL−1hydrocortisone, 10 ng mL−1EGF, 100 ng mL−1cholera 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 (pur-chased from the cell bank of Rio de Janeiro– Brazil) were also culti-vated in DMEM medium supplemented with 10 % of FBS All cultiva-tions 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 1 × 106cells/well, pipetted in a 96-wells-flat-bottomed plate The incubation process was carried out for 24 h, 37 °C, and 5 % CO2humidified incubator 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 ultra-pure water by ultrasonic treatment at 20 % amplitude (12 W cm−3) for
10 min in an ice-water bath (Wang et al., 2010)
The cytotoxic effect was evaluated using the MTT assay The cell suspension (100μL) was added to each well and incubated for 24 h, 37
°C and 5 % CO2 After, the culture was replaced by 180μL of fresh culture medium, and 20μL of the EPS solution at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg L−1were added After 24 h of incubation, 20μL of the MTT solution was added to each well to afinal concentration of 0.5
mg mL−1 Then, after 3 h of reaction at 37 °C and 5%, CO2100μL 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
Table 3 Yields, chemical analyses, and monosaccharide composition of M alpina EPS and its products of partial acid hydrolysis
Parameters Partial acid hydrolysis fractions
EPS Eluted (EF) Retained (RF)
Mw (g mol−1) c 4.9 ×
10 5
Uronic acids (%) d 0.5 nd nd
Monosaccharide composition f (mol%)
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 inFig 1D)
Trang 4replicates The following equation was used to calculate the percentage
of viability:%cell viability=(100x A595a)/A595b, where A595ais the mean
value of the treatment samples and A595b is the mean value of the
blanks
3 Results and discussion
3.1 Culture medium and optimization of EPS production
Previous work regarding culture medium optimization was carried
Fig 1 Chromatographic and spectroscopic analyses of M alpina EPS A) UV–vis scan spectrum; B) HPSEC-MALLS-RID elution profile (eluted with 0.1 M NaNO2at 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 thisfigure legend, the reader is referred to the web version of this article.)
Trang 5out 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
pro-duction was developed A 24experimental 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 thisfirst part, the first-order polynomial equations obtained from the
statistical regression for biomass production and viscosity enhancement
are as follows:
Viscosity (mPa.s) = 1.53 + 0.5*X2+ 0.23*X4– 0.1*X1X2+ 0.2*X2X4
(1) Biomass (g L−1) = 18.17 + 2.85*X1+ 1.8*X4+ 1.15*X1X4 (2)
Eq.1indicates that the increment of urea and phosphate concentrations enhanced EPS production Glucose and pH did not show a positive in-fluence 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
Fig 2 Solid-state (13C CP-MAS) NMR spectrum of EPS produced by Mortierella alpina
Fig 3 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
Trang 6regression for viscosity (mPa.s) was as follows:
Viscosity (mPa.s) = −7.83−0.002*X1 +0.162*X1 −0.117*X2
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
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−1was obtained
Fig 4 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 5 Frequency dependence of the storage and loss moduli of chitin-like EPS-based solu-tion at concentrasolu-tions of A) 5 mg mL−1, B) 10
mg mL−1and C) 15 mg mL−1of chitin in LiCl and D) 5 mg mL−1, E) 10 mg mL−1and F) 15
mg mL−1of chitin in NaCl over a wide range of temperature Storage modulus is represented in filled symbols and Loss modulus in open sym-bols
Trang 73.2 EPS characterization
3.2.1 General analyses
Physical-chemical characterization analyses of M alpina EPS and its
partial hydrolysis products are shown inTable 3 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 min centrifugation at 14,000 RPM In
the ultraviolet region, no significant absorbance of proteins was
ob-served 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-RID analysis was 4.9 × 105g mol−1(∂ =
∂
− 0.121 mL g ) n
c
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−1attributed to NH2groups, 2899 cm−1attributed 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 cm
-1 (Rumengan et al., 2014), 1574 cm−1 attributed to the NeH
de-formation of amide II, 1438 cm−1 attributed to the CH3 group
de-formation (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 NH2groups The EPS showed a higher
in-tensity band of amide I than the band of amide II, suggesting an e
ffi-cient deacetylation (Al Sagheer, Al-Sughayer, Muslim, & Elsabee,
2009)
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
bio-molecule, even at high temperatures The13C CP-MAS NMR spectrum
of the native EPS (Fig 2) showed 6 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)
Ad-ditionally, 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 the13C 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)
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 attrib-uted to C1/H1 and C2/H2 ofβ-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 corre-sponds to CH3of 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 1 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)-linkedD-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 chitin-like structure
3.3 Rheological studies
The evaluation of gel formation as a response to a temperature in-crement 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 inFig 5
Similar behavior was noted between the 0.154 NaCl and 0.28 mol
L−1LiCl solutions were used as the solvents When a concentration of 5
mg mL−1of 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−1when 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 per-iods might be needed to attain this effect This highly suggests that the kinetic of depercolation is slower than the kinetic of percolation
3.4 ABTS radical scavenging
In this study, the half-maximal inhibitory concentration (IC50) cal-culated in 5 min 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 1 min of the assay at 2.5 mg mL−1 The determined Trolox Equivalent Antioxidant Capacity (TEAC) was 989.0 μmol equiv-alent.g−1of chitin-like EPS (247 mg of Trolox.g−1of 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 phe-nomenon 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 Fig 6 % scavenging activity of EPS evaluated at 1, 5, and 10 min of reaction
time
Trang 8Fig 7 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 IC50concentrations for tumor cells
Trang 9antioxidant 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 (IC50:5 mg mL−1, 85 % of scavenging activity)
(Vinsova & Vavrikova, 2011)
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−1for
H295R, 2.1 mg mL−1for CACO-2, 1.5 mg mL−1for MDA MB 231, 1.4
mg mL−1for MDA MB 468 and 1.3 mg mL−1for MCF 07, which means
that the increment of EPS concentrations resulted in dose-dependent
cell proliferation inhibition No growth inhibition over 50 % was
ob-served in control at higher concentrations of EPS
The in vitro studies of the effect of chitin on healthy cells
demon-strated 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
in-tentionally 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
4 Conclusions
This study showed for the first time the characterization and
bioactive potential of an alternative extracellular “green” chitin
pro-duced 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 out, reaching a 50 % production increment after the
optimiza-tion process The EPS was homogeneous and had a molar mass of 4.9 ×
105 g mol−1determined by HPSEC-MALLS-RID The chitin-like EPS
structure was elucidated by different NMR techniques, especially by
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, Project administration Carlos Ricardo Soccol:
Resources, Writing - review & editing
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
Appendix A Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116716
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