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2- Department of Life Sciences Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran , hatamian_a@ut.ac.ir
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Fungi are especially popular with researchers and the scientific community because of their unique properties. Species of wild mushrooms are also grown commercially in some communities. A distinctive feature of mushrooms is that they are an important source of biologically active compounds with medicinal value that have attracted the attention of many researchers around the world (1). These physiological medicinal properties include increased levels of immunity, regulation of heart rate, improving life-threatening diseases such as cancer, stroke and heart disease. They are also a valuable source of anti-inflammatory, antioxidant, anti-cancer, probiotic, antimicrobial and anti-diabetic compounds (2). Trametes versicolor, also known as Coriolus versicolor or Polypuras versicolor in East Asian countries, has been used in traditional medicine for medicinal purposes since ancient times and is currently used in modern medicine. The fungus is found all over the world, especially in the northern hemisphere. The fungus has various bioactive components, including polysaccharide peptide, krestin polysaccharide, which have been shown to have anti-tumor and anti-cancer properties. They also protect the animal liver from aflatoxins (3). Chitin is a polysaccharide derived from the fungus Trametes versicolor, which is considered as a commercial raw material for the production of chitosan and glucosamine. Chitosan becomes a flexible and soluble polymer through the deacetylation of chitin. Chitosan is a fiber-like and homopolymeric material, first introduced as a natural cationic flocculant for wastewater treatment in 1975 and it was used industrially (4). Chitosan is also found in some fungi and has gained much attention recently due to its special properties such as different chemical structure, non-toxicity, bio-compatibility with many organs and processing in various forms such as fibers, powders, membranes, gels, sponges and fiber (5,6). These unique properties has led to its high potential to produce applied materials (7). In 2018, Mokhtari et al. Extracted and optimized chitin and chitosan of the Iranian fungus Ganoderma leucidum, for biopolymer production (9). Currently commercial chitosan is produced from marine resources, including crustaceans, but due to problems such as environmental pollution during the process of detoxification and the high cost of extraction from these resources as well as the contamination of waters with heavy metals due to the entry of petroleum products. Damage to vessels containing petroleum and ultimately contamination of hard-skinned creatures that are the main source of chitosan production have encouraged researchers with benefits such as lower costs for extraction, elimination of heavy metal contamination and mass production at the commercial level to extract this bioactive substance from Sources of fungi.
Therefore, in the present study, the production of chitin and chitosan from the native Iranian fungus Trametes Versicolor, isolated from the northern forests of Iran, was performed to extract chitosan from in vitro and extract chitin from biomass. Also, by studying the conditions of chitosan extraction, factors affecting the production of chitosan from this fungus were selected and optimized by the Taguchi method. Then chitosan FTIR analysis was performed and the chitosan deacetylation rate was calculated. Finally, the amount of chitosan antibacterial activity was calculated by disk diffusion method.
Mushroom Cultivation and Extraction of Chitin and Chitosan
Fungi were cultured in PDA (Potato dextrose agar) in vitro and kept in an incubator at 28 ° C for 7 days. Chitin and then chitosan were extracted from the biomass obtained (10). For chitin production, dried mycelium powder with 1:20 W / V ratio was mixed with NaOH 4 M and placed in a water bath at 90℃ for 3 hours. The remaining sediment is the same chitin by freeze-drying, dehydration and weight were measured (11). For extraction of chitosan, chitin was exposed to 45% concentrated sodium hydroxide for 4 hours at 90 ° C with a 1: 15 W / V ratio. The remaining sediment was washed to neutral. Finally, chitosan was dehydrated by freeze drying method and weight was measured (12).
Optimization of Environmental Variables Affecting the Process of Chitosan Separation and Production from Trametes versicolor
According to the commonly used methods for designing experiments at different levels, the Taguchi method was chosen. To find the optimum conditions for further production of chitosan, some of the effective parameters in chitosan extraction such as temperature, process time, biomass to NaOH ratio and NaOH concentration were investigated. In this study, experiments were evaluated with 4 factors at three levels. The L9 array was used for this purpose.
Factors and levels are listed in Table 1. Table 2 also shows the Taguchi L9 array.
| Factor | level | |||||
| 1- | 0 | 1+ | ||||
| NaOH (M) | A | 2 | 4 | 6 | ||
| Time (hour) | B | 1 | 3 | 5 | ||
| Temperature (℃) | C | 30 | 60 | 90 | ||
| NaOH/Biomass | D | 1.15 | 1.20 | 1.25 | ||
Table 2. Taguchi L9 array layout for chitosan production.
| Run | Conditions | |||
| NaOH | Time | Temperature | NaOH/Biomass | |
| 1 | 1 | 3 | 3 | 3 |
| 2 | 2 | 2 | 3 | 1 |
| 3 | 2 | 1 | 2 | 3 |
| 4 | 3 | 3 | 2 | 1 |
| 5 | 1 | 2 | 2 | 2 |
| 6 | 3 | 2 | 1 | 3 |
| 7 | 2 | 3 | 1 | 2 |
| 8 | 1 | 1 | 1 | 1 |
| 9 | 3 | 1 | 3 | 2 |
Characterization of Chitosan by Fourier Transform Infrared Spectrometry Analysis
Using FTIR to investigate chemical bonds and functional groups, the chitosan powder produced was prepared for FTIR test and the following equation was used to determine the degree of deacetylation of the samples (13).
(Equation1): DD=[(A1655÷A3450)]×115]
In this formula A1655 is the first type of Amide absorption peak at 1655 cm-1 as the amount of N-acetyl groups and A3450 is the Hydroxyl group (OH) absorption peak at 3450 cm-1 (14).
Evaluation of Antibacterial Properties of Chitosan
Disk diffusion method was used to evaluate the antibacterial activity of chitosan produced and two bacteria E. coli and S. aureus were studied (15).
Statistical Analysis
According to the data obtained from ANOVA in Table 4 and the results obtained from the experiments, a very high degree of similarity and agreement was observed (p<0/05). Therefore, the variables listed in Table 4 are important and effective in producing more chitosan.
After 10 days of cultivation of Trametes versicolor in PDB medium, the biomass (dry -weight) was 2.25 g/L. The obtained biomass of 0.18 gr of chitin and the obtained chitin of 0.09 gr were finally extracted. Table 3 shows the amount of chitosan produced in each experiment. The purpose of the design of the experiment was to achieve the maximum chitosan content.
Optimization of Parameters Affecting the Production of Chitosan from the Medicinal Fungus Trametes versicolor
Four factors of NaOH, time, temperature and biomass / NaOH ratios were selected to investigate the factors affecting chitosan growth. After selecting the factors, the experiments were designed and implemented using the Taguchi method in three levels with four factors. Chitosan production was then expressed in g/L.
According to the data obtained from ANOVA, a high degree of similarity and agreement is observed. Contour (C) shows a comparison chart of the results obtained from the amount of chitosan from the fungus in each experiment with data predicted by the software. As shown in Figure 1, the results of the experiments are highly consistent with the data predicted by the software, with R2 producing chitosan 0.9993 and R2 adjusted 0.9971, all of which indicate high accuracy of the experiments. Factors affecting p-value are less than 0.05. The final equation for chitosan production is:
(Equation 2): chitosan+ =0.27 -0.050 NaOH – 0/0656 Time + 0.000878 temperature + 0.0197 NaOH × Time
| Run | conditions | Response | |||||
| NaOH (M) |
Time (h) | Temperature | NaOH/Biomass | Actual chitosan (g/l) | Predicted chitosan ( g/l) | ||
| 1 | 1 | 3 | 3 | 3 | 0.12 | 0.1207 | |
| 2 | 2 | 2 | 3 | 1 | 0.135 | 0.1337 | |
| 3 | 2 | 1 | 2 | 3 | 0.137 | 0.1377 | |
| 4 | 3 | 3 | 2 | 1 | 0.23 | 0.237 | |
| 5 | 1 | 2 | 2 | 2 | 0.156 | 0.1547 | |
| 6 | 3 | 2 | 1 | 3 | 0.157 | 0.1557 | |
| 7 | 2 | 3 | 1 | 2 | 0.172 | 0.1727 | |
| 8 | 1 | 1 | 1 | 1 | 0.115 | 0.1157 | |
| 9 | 3 | 1 | 3 | 2 | 0.11 | 0.1107 | |
Table 4. ANOVA data for the variables used in the experiment
| Respond | Level | Degree of release | F-value | P-value | R2 | Obtained R² | Predicted R² |
| A-NaOH | 1 | 468.17 | 0.00211 | 0/999 | 0/9971 | 0/809 | |
| Chitosan | B-Time | 1 | 1066.17 | 0.00091 | |||
| C-Temprature | 1 | 260.04 | 0.00381 | ||||
| D- Biomass/NaOH | 2 | 198.05 | 0.0050 | ||||
| AB | 1 | 780.12 | 0.0013 | ||||
| model | 6 | 460.17 | 0.0022 |
| Chitosan (g/l) | NaOH/Biomass | Temperature (C˚) | Time (h) | NaOH (M) | The optimal value of run conditions |
| 0/261 | 1: 25 w / v | 65.6 | 4 h and 40 min | 5.94 |
FTIR Spectrum
Based on the infrared curves obtained, equation 1 degree of commercial chitosan deacetylation, 76% was obtained, and the degree of produced chitosan deacetylation was 78%.
Evaluation of the Antibacterial Activity of Chitosan Produced from Native Trametes versicolor
Antibacterial disk test was performed against Gram-positive Staphylococcus aureus and Gram-negative bacteria Escherichia coli after chitosan production from Trametes versicolor. To do this, chitosan extracted from the fungus was compressed into a tablet of the specified size. The aura created on the margin of the chitosan tablet indicates its antibacterial activity. Chloramphenicol antibiotics were used to control the test. All steps were performed with three replications. The antibacterial activity against S. aureus is higher than that of E. coli, indicating a higher chitosan efficacy than Gram-positive bacteria. The results of bacterial immunity in the disk diffusion test are presented in Table 6. Compared with chloramphenicol, S. aureus showed a greater inactivation in the application of chitosan synthesized from fungi than in E. coli.
Table 6. Bacterial non-growth rate in mm in disk diffusion test
| Staphylococcus aureus | Escherichia coli | Samples |
| 2(mm)±18 | 1(mm)±12 | Fungal chitosan |
| 3(mm)±21 | 2(mm)±23 | Chloramphenicol |
In this study, the chitosan of the medicinal fungus Trametes versicolor was optimized by the Taguchi method. The optimum conditions for NaOH concentration, time, temperature and biomass to NaOH ratio were 5.94 M, 4 h and 40 min, 65.6°C and ratio of 1 to 25 w/v, respectively. After chitin deacetylation by concentrated sodium hydroxide at 90°C, FTIR spectroscopy shows that this method had more effect on N-H, O-H, and C-H peaks. These changes in absorption indicate that alkaline and heat have eliminated the acetyl group from the chitin sample and the production of chitosan. Chitin is composed of OH, NHCOCH3, and NH2 groups and is eliminated by deacetylation so chitosan is obtained with OH and NH2 functional groups (16). Increasing the amount of alkali to 5.9 M concentration at 65°C increased protein degradation and more effective interactions on mycelium and chitin and ultimately more chitosan production.
Based on some researchers in optimizing chitin and chitosan production and comparing with the results of the present study, most of the work done on PDB medium has been used to grow fungi. In 2008, Wang et al. investigated the physical properties of chitosan fungi on Absidia coerulea, Mucor rouxii, Rhizopus oryzae, with chitosan acetylation rates of these fungi above 80% (17). This difference in the degree of deacetylation can be explained by the structure of the extracted chitosan, the degree of purity and the molecular weight of the chitosan which can be varied in other fungi. The results of bacterial Inhibition in the disk diffusion assay show that in general the gram-negative bacteria have more sequence in the membrane itself than the cell membrane layers and consequently have thicker membrane than the Gram-positive bacteria. Increased membrane strength of Gram-negative bacteria and consequently increased resistance to antibacterial agents. In 2001, Kim WJ and colleagues extracted and optimized Chitosan from the fungus Absidia coerulea, with an optimum value of 2.3 g/L (18). Also in 2009, Andipan et al. from Mucor rouxii increased chitosan production by adding molasses salt from 14.7% to 36.4% chitosan levels with deacetylation degree: 12.8% and molecular weight: 2.48×104 (19). In 2017, Abdel-Gawad and his colleagues obtained Aspergillus niger chitosan with an acetylation degree of 83.64% (14).
In 2018, Ahamed MIN and et al., investigated the production of chitosan from crab bark using sonicate waves to antibacterial activity in the resulting chitosan disc diffusion test, the growth zone diameter for S. aureus and E. coli, respectively, 12 mm and 14 mm were obtained (24). In 2019, Kulawik et al., in a review article on the role of chitosan on seafood found that higher deacetylate chitosan showed the best adsorption on Gram-negative and Gram-positive bacteria and lower pH of chitosan by bacterial cells were Improved (25).
In this study, the Iranian fungus Trametes versicolor was used and the Taguchi method was used to optimize chitosan from this fungus, which is a reason for its innovation. The results showed a high percentage of similarity of chitosan produced by fungi and commercial specimens purchased and samples in research papers. Bacterial immunity against E. coli and S. aureus in millimeters were 12±1 and 18±2, respectively, indicating a greater efficacy on Gram-positive bacteria. Chitosan extraction from fungi, optimum test conditions for NaOH concentration, time, temperature and biomass to NaOH ratio were 5.94 M, 4 h and 40 min, 65.6°C and 1:25 ratio, respectively. The chitosan concentration in this condition was 0.261 g/L.
The results obtained from a Master thesis with identification code 15730560962049 at Islamic Azad University North Tehran Branch.
Authors declared no conflict of interests.
Received: 2020/01/19 | Accepted: 2020/06/16 | ePublished: 2020/05/21
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