J Mater Sci: Mater Med (2015) 26:135 DOI 10.1007/s10856-015-5461-z

BIOMATERIALS SYNTHESIS AND CHARACTERIZATION

Isolation of chitosan from Ganoderma lucidum mushroom for biomedical applications Natali Mesa Ospina • Sandra Patricia Ospina Alvarez • Diana Marcela Escobar Sierra • Diego Fernando Rojas Vahos • Paola Andrea Zapata Ocampo • Claudia Patricia Ossa Orozco

Received: 11 September 2014 / Accepted: 9 January 2015 Ó Springer Science+Business Media New York 2015

Abstract Chitin biopolymer production and its by-product chitosan show great potential. These biomaterials have great applicability in various fields because they are non-toxic, biodegradable, biocompatible, and have antimicrobial effects. The most common source of chitin and chitosan is the crustaceous shell; however, mushrooms are an alternative source for isolating these biopolymers because their cellular wall has a high content of chitin, which may be transformed into chitosan through a deacetylation reaction. The main objective of this research was to obtain chitosan through the deacetylation of chitin isolated from the Ganoderma lucidum basidiomycetes mushroom, which is obtained through biotechnological culture. The material characterization was performed using X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, and an evaluation of cytotoxicity comparing the results obtained with results for commercial chitosan. Protocol results showed that chitosan obtained from this mushroom had a significant similitude with commercial chitosan, yet the one obtained using P2 protocol was the one that rendered the best results: including diffractogram peaks, characteristic infrared analysis bands, and an 80.29 % degree of deacetylation. Cytotoxicity in vitro testing showed that the material was non-toxic; furthermore, it rendered very promising information regarding the evaluation of future applications of this biomaterial in the field of biomedicine.

N. Mesa Ospina  D. M. Escobar Sierra  C. P. Ossa Orozco (&) Biomaterials Research Group, Bioengineering Program, Engineering Faculty, University of Antioquia UdeA, Calle 70 No. 52-21, Medellı´n, Colombia e-mail: [email protected] S. P. Ospina Alvarez  D. F. Rojas Vahos  P. A. Zapata Ocampo Biotechnology Research Group, Biology Institute, University of Antioquia UdeA, Calle 70 No. 52-21, Medellı´n, Colombia

1 Introduction Chitin is a hard, inelastic, and white polysaccharide found in the exoskeleton of insects, crabs, shrimps and lobsters, in the internal structure of other invertebrates such as prawns and insects, as well as, on the cellular wall of fungi and yeasts. It is the second most abundant biopolymer after cellulose [1]. Chitin is an n-acetyl-d-glucosamine linear polymer joined by b (1-4) glycosidic linkage: (1 ? 4, 2-acetamide-2-deoxy-b-D-glucan) and there are three different polymeric forms a, b and c [1–4]. Chitosan is a linear polysaccharide formed by randomly b-(1-4) D-glucosamine (deacetylated unit) distributed chains and n-acetyl-d-glucosamine (acetylated unit), which comes from a process known as chitin deacetylation [4]. Chemically, chitin is deemed as an inert material with a poor reactivity since it is insoluble in ordinary solvents such as water, alcohols, acetone, hexane, deluded acids, and deluded and concentrated alkalis, among others. Chitosan is at present one of most studied biopolymers because of its solubility in aqueous solutions of some acids since it shows some alkylations [4–6], and as a consequence the number of applications in various fields has increased. Chitosan is found in certain types of fungi; nevertheless, it is generally most-efficiently obtained from chitin deacetylation. Indeed, at present, publications on chitosan applications are increasing considerably. This has made chitosan a more used product than chitin, mainly due to chitosan functionality and ease of solubilization; these characteristics make it possible for chitosan to be susceptible to transformation in several forms, and it is the only natural cationic polymer [1]. It has relevant properties including antimicrobial, antifungal, and antiviral activities; it is non-toxic, biocompatible, biodegradable, emulsifying, grease-absorbent, adsorbent of contaminating metals, and

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filmogenic; therefore, it is considered that chitosan has wide-ranging applications in various industrial fields [7]. There are various techniques used to isolate chitosan reported in literature, for they mostly depend on the characteristics of the source, usually obtained in the fishing industry. However, it is possible to isolate chitosan from various algae and fungi which may be deemed good sources of chitin and chitosan. The methods used to obtain them are quite similar; nonetheless, it is necessary to use microorganism cultures to render the demineralization process unnecessary [8]. Generally, processes to obtain chitin and chitosan are conducted by means of the following consecutive stages: (1) Preparation of raw material, (2) Protein extraction (deproteination), (3) Elimination of inorganic impurities (demineralization) which is an unnecessary process in the extraction of chitin from fungi, (4) Discoloration of obtained chitin, and (5) Chitin deacetylation. The stages following the extraction of chitin are the ones involved in its conversion to chitosan, and this may be done through chemical processes or biological methods including microbiological fermentation reactions and enzymatic reactions. It is worth noting that biological methods have not been optimized yet; therefore, they do not render good yield, and they are not yet economically profitable [9]. Previous work has resulted in an optimal protocol for obtaining chitin from the fungal mycelium biomass [8]. However, due to the low solubility of the biopolymer and the need to obtain natural biomaterials for medical applications, this research has been continued in order to obtain chitosan derivative with improved solubility from fungal chitin, assessing cytotoxicity in order to determine biomedical potential. This study presents two protocols to isolate chitosan through a thermo-alkaline treatment of chitin obtained from the Ganoderma lucidum mushroom, which were carried out by varying temperatures, reagent concentrations, as well as, stirring and drying times of the sample. The chitosan obtained was evaluated on L929 cell line cultures (mouse fibroblasts) in order to validate its use as raw material in the development of implantable devices such as scaffolds, microspheres for controlled drug release, edible biofilms and applications in biomedicine.

2 Materials and methods 2.1 Microorganism and obtaining Ganoderma lucidum mycelial biomass The G. lucidum strain was cultivated in potato dextrose agar (PDA) and incubated at 26 °C for 5 days and stored at 4 °C. To activate the mushroom, 1-cm squares of mycelium were transferred to Petri dishes with MGL1 solid

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medium. These cultures were incubated between 10 and 15 days at 26 °C and stored at 4 °C [10]. The pre-inoculates were obtained taking 1-cm-diameter mycelium disks and inoculating them in 250 mL erlenmeyer flasks that had 50 mL of liquid MGL1 culture medium. These cultures were incubated at 100 rpm, 25 ± 1 °C, for 5 days [10]. A 600 mL of Pre-culture of G. lucidum was used in a 7 L stirred tank bioreactor, Bioflo 110 New BrunswickÒ, with 4.5 L of work volume of MGL1 liquid culture medium. Culture conditions in the bioreactor were 26 ± 1 °C, 400 rpm, 5.4 of pH 5 VVM of aeration (volumes of air per volume of fermenter per minute), with a light of 3.67 lmol m-2 s-1, for 3 days of culture [11]. The biomass obtained from the bioreactor was homogenized, taken to -20 °C and lyophilized at -50 °C and 25 Pa, for 72 h. 2.2 Biopolymers isolation 2.2.1 Chitin extraction Forty grams of dried biomass G. lucidum mushroom were homogenized in deionized water. The suspension was sonicated at 150 Hz. Then, the suspension was placed in a centrifuge at 7000 rpm for 15 min, and the precipitate was submitted to deproteinization with NaOH 4 M at a ratio of 1:20 (p/v), constantly stirring at 100 °C for 2 h. Later, it was washed four times using deionized water. This mixture was put into the centrifuge under the same conditions, and supernatant was eliminated. The deproteinization treatment was conducted twice. Finally, the material was dried at 50 °C until it reached a constant weight [8]. 2.2.2 Chitosan isolation Two protocols of deacetylation were developed and implemented based on reports from different authors, in order to generate a standard working protocol [6, 12–14]. The first protocol was P1, when the chitin powder was added to a NaOH solution at 45 % at a ratio of 1:10 (p/v), submitted to a temperature of 60 °C, constantly stirring at 100 rpm for 2 h. Once this time was completed, the solution was put into a centrifuge and the supernatant was discarded. After that, it was washed using deionized water. A NaOH solution at 45 % in a ratio of 1:10 (p/v) was once again added to the precipitate, at a temperature of 94 °C, constantly stirring for 2 h. Finally, it was put into the centrifuge, and the precipitate was washed using deionized water to get a neutral pH. The sample was dried in an oven at 45 °C for 4 h. The second protocol, was P2, the precipitate obtained was placed in a NaOH suspension at 50 % in a ratio of 1:10 p/v, at a 90 °C temperature and stirred at 1300 rpm for 3 h.

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After this time, it was put into the centrifuge at 7000 rpm, at 10 °C temperature and then it was once again stirred constantly in a NaOH solution at 50 % at a ratio of 1:15 (p/v) at 120 °C for 2 h. It was put into the centrifuge and the precipitate was washed using deionized water until obtaining a neutral pH, and then, the moist sample was dried in an oven at 45 °C for 4 h. 2.3 Chitosan characterization 2.3.1 X-ray diffraction (XRD) An X-Ray Diffraction analysis was performed on the chitosan powder (P1 and P2 samples for triplicate) using ´ Pert PRO MPD equipment with a 1.8 kW (40 mA y X ˚. 45 kV) ceramic Cu tube, K-alpha radiation at 1.5406 A 2.3.2 Fourier transform infrared spectroscopy (FTIR) FTIR spectra for P1 and P2 samples, in triplicate, were obtained using a Spectrum One spectrophotometer that had a DTGS Perkı´n ElmerÒ detector. For this analysis, samples were dispersed in KBr to form the pellets that were to be tested. The analysis was conducted at a temperature of 24 °C, and eight scannings were conducted with a (m) 4000–450 cm-1 wavenumber range and a 4 cm-1 resolution. 2.3.3 N-acetylation percentage (DA) The deacetylation percentage of the sample was obtained with the FTIR spectrum. This spectroscopic technique allows determining the percentage of N-acetylation through the correlation of some absorbance bands, which involve some amide bands (I–III) and another band like an internal reference to correct the thickness of the (KBr) potassium bromide pellet used. Brugnerotto et al. [15] used the amide III band located at 1320 cm-1 and as a reference the band of the methyl groups at 1420 cm-1; with this, linear correlation the deacetylation DA (%) was obtained, Eqs. 1 and 2.   A1320 Nacetylation ð%Þ ¼ 31:92  12:2 ð1Þ A1420 DA ð%Þ  100  Nacetylation ð%Þ:

ð2Þ

2.3.4 Thermogravimetric analysis (TGA) This test was performed in an air atmosphere with a TGA Q500 device at a 10 °C/min warming speed, and the sample was warmed up at temperature intervals ranging from 0 to 900 °C. A thermogravimetric analysis was performed only on the material obtained using P2 process.

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2.3.5 Cytotoxicity in vitro These studies of biocompatibility were performed following the method reported in the United States Pharmacopoeia (USP29) [16]. A cytotoxic test was carried out only on chitosan obtained using P2 process. An elution test was conducted to evaluate the effect of chitosan powder extract on L929 cell line cultures (mouse fibroblasts), when the monolayers had reached 80 % confluence, extract was added to the culture medium, and the cells were incubated at 37 °C with 5 % CO2 for 24 h. The estimate of the results was based on the morphological appearance of cells after incubation. 2.3.6 Sample preparation 0.2 g of chitosan powder were macerated in 6 ml of an RMPI 1640 Glutamax culture medium supplemented with 10 % fetal bovine serum (FBS) at 37 °C, for 24 h. A nonreactive ultra-high molecular weight polyethylene was used as a negative control; a fragment of sterile latex condom fragments without any lubricant was used as a positive control [17]. 2.3.7 Culture preparation L929 cell line cultures (mouse fibroblasts) were performed under standard conditions at 37 °C in a wet atmosphere with 5 % CO2, growing the cells at a 1 9 105 cell/mL density for 24 h in an RMPI 1640 Glutamax medium and 10 % SBF. For adherent cell subcultures, the medium was removed from the bottles, and it was washed with PBS. Then, cells were detached using 1 mL of trypsin (0–25 %), letting it act for 3 min. After, 5 mL of complete culture medium were added to neutralize the trypsin; the cell suspension was collected, and it was put into the centrifuge for 5 min at 1200 rpm. Once the cell buttons were obtained, they were re-suspended in a complete culture medium, and cellular density was adjusted to a 1 9 105 cells/mL. Later, cells were grown in 2 mL Petri dishes of this suspension to obtain a monolayer. After incubating at 37 °C in a wet atmosphere and 5 % of CO2, a blue 300-lL trypan solution (0.01 %) was added to each well. Then, it was incubated at 37 °C in a wet atmosphere and 5 % CO2 for 2 min. Finally, cell viability and morphology were verified. Cells were microscopically evaluated and their reactivity was classified according to the parameters reported in Table 1. A sample complies with toxicity requirements, only if there is low reactivity (two grades); and if, an appropriate response is observed in the controls.

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3 Results and discussion

Table 2 Production of biomass and polysaccharides under 7L bioreactor conditions

3.1 Biomass production and the obtaining of chitosan A 7 L bioreactor level culture has allowed knowing and analyzing the behavior of G. lucidum in these controlled systems, forms and growth times, and its special medium needs. This made it possible to reduce the source of carbon concentration (from 50 to 20 g/L), and the general result was to obtain a new culture protocol achieving promising results including raw materials to develop new products in terms of biomass. Nonetheless, this requires an upgrade in terms of obtaining chitin and chitosan (Table 2). Pochanavanich et al. [18], obtained chitosan from different fungus (Aspergillus niger TISTR3245, Rhizopus oryzae TISTR3189, Lentinus edodes no. 1, Pleurotus sajocaju no. 2, Zygosaccharomyces rouxii TISTR5058 and, Candida albicans TISTR5239). They carried out cultures fungus during different periods among 15–21 days, and then, they evaluated biomass and chitosan production yield. Rhizopus oryzae TISTR3245 was shown to give a maximal yield of chitosan at 138 mg g-1 dry weight, this value was obtained for 6 days of culture. In this research the fungi was cultivated for 3 days, and it was obtained 83.23 ± 4.53 mg of chitosan/g of biomass; this values are approximate to obtain for Aspergillus niger TISTR3245 and Rhizopus oryzae TISTR3189 for 3 days of culture; this fact proved than the process is efficient, due to above, it is comparable with other fungi. Nevertheless, there are many factors important which the chitosan content of fungi than depends on fungal strains, mycelial age, cultivation medium and conditions; and the chitosan content of fungal mycelia than also depends on the chitosan extraction method. 3.2 Chitosan characterization 3.2.1 Sample morphology Chitin and chitosan samples are formed by small flakes which vary in size, and chitin powder is smaller and more homogeneous. Furthermore, chitin obtained following the

Biomass (g/L)

19.72 ± 1.06

Productivity (g biomass/day)

6.57

Chitosan (mg of chitosan/g of biomass)

83.23 ± 4.53

protocol in Ospina et al. [8] is light brown because there has not been a discoloration process. This color is perhaps due to the fact that there is still lignin in it. This component gives the woody color to certain types of vegetables, and fungi, including Ganoderma [19, 20]. This species is characterized for its great ligninolytic potential [20]. This polymer is insoluble in acids, but it is soluble in strong alkalis like NaOH, which was added in the deacetylation process. Nevertheless, chitosan is beige in colour because of chitin deacetylation processes and washings lead to lighter color material; hence, color intensity proportionally depends on the number of washes it has undergone. 3.2.2 X-ray diffraction (DRX) Figure 1 shows the chitosan diffractogram obtained using P1 and P2 protocols, (a and b) and commercial chitosan (c), it could be observed that chitosan obtained using P1 shows characteristic peaks at 2h = 9.2° and 20°, and some peaks with lower intensity at 2h = 25.4° and 30.2°. On the other hand, chitosan obtained using P2 shows characteristic peaks at 2h = 10° and 20°. The influence of temperatures and NaOH concentrations in the crystalline structure in these diffractograms could also be noticed. These results are coherent with commercial chitosan, which has characteristic peaks found at 2h = 9.0° and 19.7°; this shows the similarity of both components and suggests that the samples obtained using the two protocols are chitosan. Nonetheless, the one obtained using P2 is better, as it has fewer impurities and more crystallinity. This shows that when the deacetylation temperature, the NaOH concentration, and the washing time are increased, the sample purity is also increased. Likewise, Ferna´ndez Cervera et al. [21] observed a similar result for two stages

Table 1 Grade, reactivity, and conditions of cultures Grade

Reactivity

Conditions of cultures

0

None

Discrete intracytoplasmic granules; no cell lysis

1

Slight

Not more than 20 % of the cells are round, loosely attached, and without intracytoplasmic granules; occasional lysed cells are present

2

Mild

Not more than 50 % of the cells are round and devoid of intracytoplasmic granules; no extensive cell lysis and empty areas between cells

3

Moderate

Not more than 70 % of the cell layers contain rounded cells or are lysed

4

Severe

Nearly complete destruction of the cell layers

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Fig. 1 Chitosan diffractograms obtained from Ganoderma lucidum fungi using P1 and P2 protocol and commercial chitosan

Fig. 2 FTIR comparison of a standard chitosan sample and chitosan obtained from Ganoderma lucidum fungi using P1 and P2

of deacetylation including a first one with NaOH 45 % at 130 °C for 30 min, and then a second one with NaOH 49 % under the same conditions of time and temperature used at the beginning when peaks at 2h = 10° and 20° were reported.

P1 and 1425 cm-1 for P2 as reference for the linear correlation expressed in Eq. 1 were used; an 80.14 % percentage of deacetylation for protocol P1 and 80.29 % for P2 were obtained. To obtain a soluble product, chitosan must have a degree of deacetylation between 80 and 95 %. Generally, chitosan has between 70 and 95 % of deacetylation; for 100 % percentages of deacetylation, the polymer transforms into a product known as chitan [23]. P1 and P2’s percentage of deacetylation is over 80 %, and it is similar to found by Pochanavanich et al. [18] who found that the degree of deacetylation of fungal chitosan was 84–90 % and Wu et al. [24] who obtain a degree of deacetylation 80.5 % for chitosan from Mucor rouxii. Furthermore, this value found within the range reported for products with adequate solubility was also greater than the percentage of deacetylation of commercial chitosan used for comparison, which was 75 %. Chitosan with a high degree of deacetylation has high positive charges and is more suitable for food applications as a coagulating or chelating agent, a clarifying agent or an antimicrobial agent [25].

3.2.3 Fourier transform infrared spectroscopy (FTIR) Figure 2 shows the FTIR spectrum of chitosan samples obtained using protocols P1 and P2. These spectra are also compared to a sample of commercial chitosan. The correlation rendered for the equipment between the chitosan obtained from the fungi using P1 and standard chitosan was 79 %; whilst for chitosan obtained using protocol P2, the correlation rendered was 83 %. Table 3 compares the values of characteristic bands for each chitosan spectrum obtained from the mushroom using P1 and P2, standard chitosan, and a spectrum reported in literature [3]. Characteristic chitosan bands for amide I in approximately 1630 cm-1 for protocols P1 and P2, for NH2 in 1561 cm-1, and the band at 1318 cm-1 which corresponds to amide III can be observed. 3.2.4 N-acetylation percentage (DA) The infrared technique was used for a qualitative or quantitative evaluation of DA %, determining absorption coefficients. In all these determinations using FTIR, it is very important to adequately select the baselines to calculate absorbance in order to obtain reliable results [15, 22]. To evaluate N-acetylation percentage of the samples obtained using protocols P1 and P2, implemented in this study, characteristic bands, the amide III located at 1317 cm-1 for P1 and 1318 cm-1 for P2, methyl groups at 1402 cm-1 for

3.2.5 Thermogravimetric analysis (TGA) Since previous results showed P2 as the most efficient protocol, a thermogravimetric analysis was performed only on the material obtained using this process. The result of the TGA analysis of chitosan is shown in Fig. 3. The curve shows evidence of a loss of weight in three stages. The first stage of 13.61 % weight loss occurs in the range between 10 and 150 °C; this could be a result of the loss of absorbed water. The second stage starts at approximately 150 °C and continues to 350 °C, and during this interval there is a 48.08 % weight loss. Chitosan starts degrading at 273.2 °C, so it is only safe to work with this material up to

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Table 3 Comparison of FTIR characteristic bands for obtained chitosan and chitosan patterns Molecule

Chitosan reported in literature [4] (cm-1)

Standard Chitosan (cm-1)

Chitosan obtained from the G. lucidum mushroom using P1 (cm-1)

Chitosan obtained from the G. lucidum mushroom using P2 (cm-1)

Group tension –OH

3450

3445

3445

3448

Group tension N–H

3292







Group tension C–H

2919 and 2868

2925 and 2897

2927 and 2873

2925 and 2895

Amide I

1655

1660

1635

1640

Tension CH3

1430

1440

1402

1425

Doubling the Group –NH2

1550

1590



1560

Amide III

1313

1312

1317

1318

Antisymmetric tension bridge C–O–C

1154

1160

1158

1160

Tension C–O

1080 and 1029

1073 and 1029

1080 and 1031

1079 and 1052

Anomeric group CH tension

896

890

898

900

Fig. 3 TGA Analysis of a chitosan sample obtained using P2

this temperature. In the third stage, there is a 38.03 % weight loss in a temperature range of 350–450 °C; these two last weight losses are due to saccharide degradation in the molecular structure of organic material and to a further final degradation of organic material. The aforementioned can also be corroborated with a final residual percentage of 0.1372 %, since there is no presence of organic matter at temperatures over 500 °C; this indicates that no metal or inorganic substances exist in the biopolymer; that may affect the possible chitosan medical applications. The TGA curves indicated that chitosan obtained from G. lucidum fungus is thermally stable like the chitosan obtained from other sources; those such as Ramya et al.

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[26] and Prˇichystalova´ et al. [27] found a degradation peak at 300 °C approximately for chitosan obtained from crab shells and Paulino et al. [28], obtained a first degradation peak at 250 °C for chitosan produced from chrysalides. Kaya et al. [29] obtained chitosan from Colorado potato beetle (Leptinotarsa decemlineata), the temperature at which the maximum degradation of chitosan occurred was 289 °C for the chitosan extracted from the adults and 292 °C for the chitosan extracted from the larvae, this values are similar to obtain from G. lucidum chitosan. The Sigma Aldrich commercial chitosan was studied by Zakaria et al. [30] who found a degradation peak at 280.82 °C, and a final residual percentage of 32.7 % and

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Fig. 4 Analysis of the cytotoxicity in vitro of chitosan obtained for P2 using an elution method

commercial chitosan obtained from Fluka studied by Liao et al. [31] had the maximum degradation peak at 313 °C, with a final residual mass of 37.14 %; both commercial chitosan were isolated to crustaceous and their residual mass remained is highest than chitosan from G. lucidum fungus. As mentioned above, crustaceous are commonly used for chitosan extraction but the high content of minerals make than there are residues that are difficult to eliminate in isolation processes and causes effects on process efficiency.

It is possible to conclude that G. lucidum mushroom is a potential chitosan source with advantages, namely, fungi´s high growth rate, low content of minerals, and the possibility that their waste can be used in both food and paper industries [32]. 3.2.6 Cytotoxicity in vitro The results show that the obtained biomaterial is adequate, since they did not show incompatibility with cell growth. It

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can be noticed that cells which remained in contact with the chitosan extract were attached forming a confluent monolayer without any cytological alteration (Fig. 4). The cell viability technique with trypan blue showed that a cell viability loss cannot be observed either in treated cells in contact with chitosan or in the negative control (ultra-high molecular weight polyethylene); this is different from what happened to the positive control (a latex condom fragments without any lubricant), in which all the cells detached from the plate and died [17]. Bearing in mind Table 1 as the base, chitosan reactivity grade is 0, which indicates that the cells after incubation show discrete intracytoplasmic granules, and that there is no cell lysis. This result is favorable for the use of biomaterial in various reported applications for cosmetic, pharmaceutical, biomedical, and food industries [33–36].

4 Conclusions Chitosan was obtained from a G. lucidum mushroom in two stages using two protocols (P1 and P2), in which P2 was the protocol used to obtain biomaterial having the desirable characteristics in terms of DRX, FTIR, percentage of deacetylation and a Thermogravimetric Analysis. The DRX showed peaks which were very defined and characteristic from about 10°–20°, similar to the ones reported in literature with a smaller number of impurities and a greater percentage of the crystalline phase. The FTIR analysis showed that chitosan obtained using protocol P2 has a greater correlation value in comparison to standard chitosan. The percentage of deacetylation was 80.29 %, and the thermogravimetric analysis showed that the material begins to degrade at 273.2 °C, which is similar to the temperature reported in literature. Finally, chitosan powder fulfills bioreactivity in vitro requirements, since it obtained grade 0 (no reactivity), and this indicates that the material is biocompatible. All above mentioned characteristics are fundamental and promising characteristics for chitosan use in specialized sectors such as biomedicine, pharmaceutics, cosmetics and food, among many others. Acknowledgments The authors of this study express their gratitude to both Universidad de Antioquia CODI for its funding of this research project and to the Biotechnology and Biomaterial Research Groups.

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Isolation of chitosan from Ganoderma lucidum mushroom for biomedical applications.

Chitin biopolymer production and its by-product chitosan show great potential. These biomaterials have great applicability in various fields because t...
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