International Journal of Biological Macromolecules 47 (2010) 180183

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International Journal of Biological Macromolecules

Production, physiochemical and antimicrobial properties of fungal chitosan from Rhizomucor miehei and Mucor racemosus
Farzaneh Tajdini a, , Mohammad Ali Amini a,b , Nastaran Nassi-Varcheh c , Mohammad Ali Faramarzi d
a

Department of Microbiology, Faculty of Veterinary Medicine, Islamic Azad University, Karaj Branch, Karaj, Iran Young Researchers Club, Islamic Azad University, Karaj Branch, Karaj, Iran Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran d Department of Pharmaceutical Biotechnology, Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran
b c

a r t i c l e

i n f o

a b s t r a c t
Chitosan was isolated and puried from the mycelia of Rhizomucor miehei and Mucor racemosus. To characterize the extracted materials, their FTIR spectra were compared with that of shrimp chitosan. Final degree of deacetylation which determined by 1 H NMR was obtained as 98.6% for chitosan from R. miehei (named as C1) and 97.1% for chitosan from M. racemosus (named as C2), respectively. To investigate the antimicrobial properties of the isolated fungal chitosans, minimum inhibitory concentration (MIC) values were performed against twelve strains of bacteria and fungi. Data obtained generally showed that the antibacterial and antifungal activities of the puried fungal chitosans were more effective against Escherichia coli, Pseudomonas aeroginosa, Candida albicans and Candida glabrata in comparison to the shrimp chitosan. Taken together, the results suggest that the use of the fungal chitosan could be of interest as a suitable alternative source to shrimp chitosan. 2010 Elsevier B.V. All rights reserved.

Article history: Received 11 January 2010 Received in revised form 3 May 2010 Accepted 5 May 2010 Available online 13 May 2010 Keywords: Fungal chitosan Characterization Antibacterial activity Antifungal activity

1. Introduction Chitosan, the deacetylated derivative of chitin, is a naturally polymer of -1,4-glucosamine. It is nontoxic, biodegradable and particularly abundant in the cell wall of crustaceans and microbial biomasses. Chitosans with various molecular weights and degrees of deacetylation have been found wide spectra of applications such as bactericidal [1] and antitumor agents [2], carriers in DNA [3] and drug delivery systems [4], adjuvant for vaccine delivery [5] and articial skin bases [6]. Antibacterial and antifungal activities of chitosan have been widely investigated and its feasibility as natural antimicrobial agents has been proved throughout several studies [79]. Due to its biodegradable and antimicrobial properties, chitosan is applied in food and pharmaceutical industries [1012]. It is also indicated that inhibitory effect of chitosan on the growth of bacteria varies according to its degree of deacetylation and functional groups [13,14]. The positive charge of chitosan, which favors interaction with the anionic charges of yeasts plasma membrane, is the reason of its antifungal activities [15]. Antibacterial activity of fungal chitosan has been shown only in a few reports [16,17]. Jeihanpour and coworkers [18] also showed that the possi-

ble mechanism for antibacterial activity of fungal chitosan could be the disruption of the outer membrane but not preventing the nutrients from entering into the cells. Although chitosan is commercially produced from shrimp, lobster and crab shell chitin deacetylation, this is a limited and seasonal-dependent supply. Chitosan could be also found in the cell walls of fungi families such as Zygomycetes and Basidiomycetes [1921]. The fungal approach has benet of easy handling, harvesting and control to produce high quality chitosan; and different types of chitosans produced by diverse strains with individual environmental and nutritional conditions [22,23]. The objective of this study was to evaluate Rhizomucor miehei and Mucor racemosus as new sources for chitosan extraction. The obtained chitosans were characterized and their antimicrobial properties were also studied. Despite the extensive researches on antifungal activity of crustacean chitosan [24,25], to the authors knowledge, there is no such study for fungal chitosan.

2. Materials and methods 2.1. Materials Chitosan from shrimp shell (MW < 18 kDa, degree of deacetylation = 96.9%) was kindly gifted from Eastar Holding Group (China). All other chemicals were of analytical grades and obtained from Merck (Darmstadt, Germany).

Corresponding author. Tel.: +98 261 4402251; fax: +98 261 4402251. E-mail address: [email protected] (F. Tajdini). 0141-8130/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.05.002

F. Tajdini et al. / International Journal of Biological Macromolecules 47 (2010) 180183 Table 1 The characteristics of extracted chitosan from R. meihei and M. racemosus. Origin of preparation R. meihei M. racemosus
a

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Fungal dry biomass at 7 days (g1 ) 4.1 3.8

Chitosan yield (%) 13.67 11.72

Initial DDa (%) 80.6 84.4

DDa after deacetylation procedure (%) 98.6 97.1

Ash (%) 0.94 0.84

DD: deacetylation degree.

2.2. Microorganisms Rhizomucor miehei ATCC 26282 and a soil isolate of M. racemosus [26] were used for chitosan extraction experiment. Gram-positive (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228) and gramnegative (Escherichia coli ATCC 8739, Klebsiella pneumonia ATCC 10031, Pseudomonas aeroginosa ATCC 9021) bacteria were applied for evaluating the antibacterial activities of fungal chitosans. Investigation on the antifungal activity of the extracted chitosans was performed using fungal strains (Saccharomyces cerevisiae NCYC 694, Saccharomyces cerevisiae ATCC 9763, Candida glabrata ATCC 90030, Candida albicans ATCC 10231 and two clinical isolates of Candida albicans S1 and S2).

2.6. Infrared spectra Approximately 23 mg of shrimp chitosan, C1 and C2 was mixed with 100 mg of potassium bromide. Forty milligrams of the mixture was used to prepare KBr pellet. Infrared spectra were recorded on a NEXUS-FTIR 870 ThermoNicolet FTIR spectrometer. 2.7. Determination of deacetylation degree (DD) The extracted chitosans were individually dissolved in D2 O/triuoroacetic acid (1000:1; v/v) solution and 1 H NMR spectra were recorded using the FTNMR Varian Unity Plus spectrometer at 400 MHz. 2.8. Antimicrobial activity assessments Fungal chitosan solutions were prepared in 0.5% acetic acid and autoclaved at 121 C for 15 min. After adding 100 l of Muller Hinton broth (MHB) or Sabouraud dextrose broth (SDB) into a 96-well microplate for bacteria or yeasts, respectively, an amount of 100 l of each chitosan solution was added to the wells to get the nal concentrations of 1%, 0.8%, 0.6%, 0.4%, 0.2%, 0.1%, 0.05% and 0.025% (w/v). For preparation of the microbial suspension, all bacteria and yeasts were inoculated on Muller Hinton agar (MHA) and Sabouraud dextrose agar (SDA) and incubated at 35 C for 24 h. After incubation, the microbial suspensions were prepared in 0.9% NaCl solution (w/v), and then diluted by MHB or SDB to adjust the concentration of bacteria and yeasts as about 2 108 and 106 (CFU/ml), respectively. An amount of 100 l of each suspension was put into the wells and mixed with the respective testing substance. All microplates were incubated for 24 h at 35 C. Similar procedure without the addition of chitosan was carried out as blank. All experiments were performed in duplicate and the average values were reported. 3. Results and discussion The characteristics of chitosans isolated from R. miehei (C1) and M. racemosus (C2) are presented in Table 1. The higher biomass and yield of chitosan was obtained from of R. miehei. Obtained data are comparable with the previous studies [16,30,31]. Chitosans yield from this study were higher than those of Zygomycetes strains reported by Tan et al. [32] and Silva Amorim et al. [33]. 3.1. FTIR and 1 H NMR characterizations As illustrated in Fig. 1, the FTIR spectra of C1 and C2 were similar to those of the shrimp chitosan and other extracted fungal chitosans [21,23]. The peak values of 1651 and 1599 cm1 were carbonyl stretching c = 0 (amide I) and amine bending NH (amide II), respectively [34]. The absorption band at 1324 cm1 could be attributed to the amide III band. The band at 898 cm1 was referenced as the -anomer [35]. The structures of acetylated and deacetylated monomers of C1 and C2 were characterized by 1 H NMR spectroscopy. Fig. 2 shows

2.3. Culture conditions for chitosan production Inoculates were separately prepared from the sporangioles of R. miehei and M. racemosus harvested from the cultures grown in a 7-day cultivation at 30 C on Petri dishes containing Potato dextrose agar (PDA). Spore suspensions were prepared and adjusted to 107 sporangioles/ml using a hematocytometer for counting. For submerged cultures, 10 ml of the inoculums were individually cultivated into 290 ml of Sabouraod dextrose broth (SDB) and incubated at 28 C for 7 days in a rotary shaker at 120 rpm.

2.4. Chitosan extraction Mycelia from each fungus were harvested by ltration, washed twice with distilled water and dried using lyophilization. Dry fungal mycelia were treated under NaOH 10% (1:30; w/v) at 121 C for 20 min. Alkali insoluble fraction was centrifuged at 12,000 g for 15 min, washed with distilled water to a neutral pH and recentrifuged. The precipitate was suspended in acetic acid 5% (1:40; w/v) at 95 C for 8 h, closely following the methods of Crestini et al. [27] and Pochanavanich and Suntornsuk [28]. The acid insoluble components were removed after centrifugation at 12,000 g for 15 min and the supernatant was then separated. The chitosan was precipitated by adjusting the pH up to 10 with 2 M NaOH, then ltered, washed with distilled water and acetone and dried at room temperature.

2.5. Purication and deacetylation of chitosan Purication and deacetylation of chitosan was performed using a modied method of Di Mario et al. [21] and Tan et al. [29]. Briey, extracted fungal chitosan, 0.5 g, and 20 ml of NaOH 45% (w/v) were placed into a 500 ml-round bottom ask. An amount of 0.05 g of NaBH4 was added to the mixture and the suspension was then reuxed, while stirring, at 95 C under nitrogen atmosphere for 2 h. After centrifugation at 12,000 g for 15 min, the precipitate was washed by hot water (80 C) to be neutralized and was then lyophilized. The alkali treatment was performed twice. The obtained chitosans from R. miehei and M. racemosus were coded as C1 and C2, respectively.

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F. Tajdini et al. / International Journal of Biological Macromolecules 47 (2010) 180183 Table 2 MIC values of fungal and shrimp chitosans against six strains of bacteria (%). Bacteria Escherichia coli ATCC 8739 Pseudomonas aeroginosa ATCC 9021 Klebsiella pneumonia ATCC 10031 Staphylococcus epidermidis ATCC 12228 Staphylococcus aureus ATCC 6538 Bacillus subtilis ATCC 6633 C1 0.1 0.2 0.05 0.05 0.05 0.1 C2 0.1 0.2 0.1 0.05 0.1 0.2 Shrimp chitosan 0.2 0.4 0.05 0.05 0.1 0.1

of both monomers. The deacetylation degrees (DD) of CS1 and CS2 were calculated by the following equation [36]: DD = 1 (1/3)HAc (1/6)H26 100

In the above equation, H-Ac is represented to area under curve (AUC) of the peak at 1.82 ppm and H2-6 is represented to AUC of the H2-6 in glucose skeleton (the peak at 3.24 ppm). The initial DD values of C1 and C2 were 80.6% and 84.4%, respectively, which increased to 98.6% and 97.1% after the purication and deacetylation procedure. 3.2. Antibacterial activity of fungal chitosans
Fig. 1. FTIR spectra of shrimp chitosan (a) and chitosans from R. meihei (b) and M. racemosus (c).

the 1 H NMR spectra of chitosan from M. racemosus before (Fig. 2a) and after (Fig. 2b) purication and deacetylation procedures. Peaks at 1.82 and 2.9 ppm were referenced as acetyl group (H-Ac) and deacetylated monomer of H2, respectively. The peak at 3.24 ppm was the signal from protons H2, H3, H4, H5, H6, and H6 (H2-6)

Antibacterial activity of the chitosans was investigated against six bacteria and dened as minimum inhibitory concentration (MIC) value (Table 2). All chitosans were more effective to inhibit the growth of gram positive bacteria than gram negatives. In comparison to shrimp chitosan, fungal chitosans, showed higher inhibitory effects, especially on E. coli and P. aeroginosa. An amount of 0.1% concentration of fungal chitosans blocked the growth of all selected gram negative bacteria except P. aeroginosa. Among gram negative bacteria, lower concentration of C1 and shrimp chitosan was completely stopped the growth of K. pneumonia. S. epidermidis was inhibited at chitosan concentration of 0.05% as similarly reported by Jeon et al. [37], which showed more sensitivity to chitosan than other bacteria. Our study showed that chitosans were more effective against S. aureus and S. epidermidis than B. subtilis. In spit of applying 0.05% concentration of C1, 0.1% concentration of shrimp chitosan was needed to suppress the growth of S. aureus. According to No and coworkers experiments on the inhibitory effect of chitosan for E. coli and S. aureus [38], MIC values were 0.1% which is comparable to, or lower than our current result. Jeihanpour and coworkers [18] has also reported that E. coli, K. pneumonia and S. aureus were more sensitive to crustacean chitosan than fungal chitosan. These differences may be attributed to various experimental methods, chitosan sources and characterization, deacetylation method [39] and chitosan solvents [40]. The comparison between the MIC values of examined chitosans revealed that C1 could be a more preferable antibacterial agent. All bacteria were grown in blanks containing acetic acid 0.5% without chitosan. 3.3. Antifungal activity of fungal chitosans Antifungal activity of fungal origin and shrimp chitosans were assessed by determination of their inhibitory effects on the growth of C. albicans, C. glabrata, two clinical strains of C. albians and two strains of S. cerevisiae by the following concentrations of 1%, 0.8%, 0.6%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025% (w/v). As illustrated in Table 3, the MIC values of fungal chitosans for C. albians and C. glabrata were lower than those for the shrimp chitosan. The growths of all Canddida spp. were blocked by 0.05% concentration of fungal chitosan, except C2 for Clinical C. albicans S1. Similar to

Fig. 2. 1 H NMR spectrum of chitosan from M. racemosus before (a) after (b) purication and deacetylation procedures.

F. Tajdini et al. / International Journal of Biological Macromolecules 47 (2010) 180183 Table 3 MIC values of fungal and shrimp chitosans against six strains of fungi (%). Fungi Candida albicans ATCC 10231 Candida glabrata ATCC 90030 Clinical isolate Candida albicans S1 Clinical isolate Candida albicans S2 Saccharomyces cerevisiae NCYC 694 Saccharomyces cerevisiae ATCC 9763 C1 0.05 <0.025 0.05 0.05 0.1 0.2 C2 0.05 <0.025 0.05 0.1 0.05 0.1 Shrimp chitosan 0.1 0.05 0.05 0.05 0.1 0.2

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the result of Seyfarth et al. [25], C. glabrata were more sensitive to the chitosans, especially the fungi-originated ones. The resistance of both strains of S. cerevisiae to the chitosans was more than all strains of Candida spp. The same results have also been reported by Roller and Covill [41]. Data showed higher inhibitory effect of C2 than other studied chitosans. Obviously, the exposure time of yeast to chitosan during incubation could be an important factor on the calculated MIC value [8]. In all blank cultures, yeasts were grown and it was concluded that acetic acid 0.5% had no inhibitory effect. 4. Conclusion In this study, the chitosans with high degree of deacetylation (above 97%) were extracted from R. miehei and M. racemosus. DD of chitosan has been introduced as an effective factor on its antimicrobial activity. Considering the close similarity of DD in the fungal chitosans to shrimp chitosan, their observed differences in antimicrobial activity, especially against C. albicans, C. glabrata, E. coli and P. aeroginosa might be attributed to the chitosan origin. It is also notable that in contrast to the applied simple and fast method described in this paper for extracting pure fungal chitosan, usual extraction of chitosan from crustaceans requires higher concentrations of sodium hydroxide at high temperature and, in general, the whole procedure is more costly and time consuming. Therefore, the applied fungi could be considered as a good alternative source of chitosan. Acknowledgments This work was nancially supported by the grant No. 3042 from Islamic Azad University, Karaj Branch, Karaj, Iran. The helpful advice of Dr. Mohsen Amini (Tehran University of Medical Sciences) is also acknowledged.