Introduction and methods

Chitin, the insoluble linear b1,4- linked homopolymer of N-acetyl-D-glucosamine (GlcNAc), is the second most abundant natural polysaccharide (after cellulose). Chitosan is a cationic amino polysaccharide, essentially composed of b-1,4 D-glucosamine (GlcNAc) linked to N-acetyl-D-glucosamine residues (Andrade et al. 2000; Campos-Takaki, 2005), derived from de-N-acetylation of chitin (Tharanathan and Kittur, 2003; Amorim et al. 2005). These polysaccharides are found in a wide range of natural sources, such as crustaceans, insects annelids, mollusks, coelenterates and is a common constituent of fungal cell walls (Andrade et al. 2003; Synowiecke and Al-Khatteb, 2003; Franco et al. 2005).

Chitin and chitosan hold a great economic value as due to their versatile biological activities and chemical applications, mainly in medical (Yadav and Bhise, 2004) and pharmaceutical (Takeuchi et al. 2001; Kato et al. 2003) areas. The wide range of applications of these polymers has been extensively studied (Yadav and Bhise, 2004; Campos-Takaki, 2005). Chitin and chitosan show peculiar properties, such as: biodegradability, biocompatibility, bioactivity, selective permeability, polieletrolic action, chelation, ion exchange properties, antitumor and antimicrobial activity (Dos Santos et al. 2003; Yadav and Bhise, 2004), and adsorption capacity (Tharanathan and Kittur, 2003; Franco et al. 2004).

Recent advances in fermentation technologies suggest that the cultivation of selected fungi can provide an alternative source of chitin and chitosan. The amount of these polysaccharides depends of the fungi species and cultural conditions (Tan et al. 1996; Pochanavanich and Suntornsuk, 2002; Andrade et al. 2003; Synowiecke and Al-Khatteb, 2003). The use of biomass from fungi has demonstrated great advantages, such as: independence of seasonal factor, wide scale production, and simultaneous extraction of chitin and chitosan. The extraction process showed here a resulting alternative product in reduction of the time and cost required for production, and at least, the absence of contamination. In that case, the contamination consisted in mainly the proteins that could cause allergy reactions from shellfish (Andrade et al. 2000; Amorim et al. 2001; Nadarajah et al. 2001; Andrade et al. 2003; Franco et al. 2005). However, the production of chitin and chitosan from fungi, it's usually used complex or synthetics cultures media, which are expensive. It’s becomes necessary to obtain economic culture media that promote the growth of fungi and stimulate the production of the polymers.

On the other hand, Yam bean (Pachyrhizus erosus L. Urban) is a leguminous plant native from the Amazon region and Mexico semiarid region (Sorensen, 1996). The leguminous produces comestible tubercles and seeds with high level of protein and lipids. The tubercles were used as a good starch source for several industrial purposes (Sarangbin and Watanapokasin, 1997; Stamford et al. 1998; Stamford et al. 2001). The main characteristic of yam bean is the simple manipulation and low nutrition requirements when compared with other similar tuberous roots. The yields are superior to 60 t/ha (Stamford et al. 1998; Stamford et al. 2001; Stamford et al. 2003).

The present paper aims to investigate chitin and chitosan production by Cunninghamella elegans (UCP 542), grown on submerse fermentation using yam bean as economic culture medium compared  with traditional culture media.

Cunninghamella elegans UCP 542 isolated from mangrove sediment situated in Rio Formoso, PE, Brazil was maintained at 4ºC on Potato Dextrose Agar (PDA) slants. C. elegans was grown, for chitin and chitosan production, in five different culture media: a) Sabouraud sucrose- (bacteriological peptone (10 g) and sucrose (20 g) per litre of distilled water, pH 5.7); b) Hesseltine and Anderson- (glucose (40 g); asparagine (2 g); chloridrate of thiamine (0.05 mg); potassium phosphate (0.50 g) and magnesium sulphate (0.25 g) per litre of distilled water, pH 5.2); c) Andrade et al (2000)- (glucose (60 g); asparagine (3 g); chloridrate of thiamine (0.08 mg); potassium phosphate (0.50 g) and magnesium sulphate (0.25 g) per litre of distilled water, pH 5.1); d) Malte glucose- (Yeast Malte (17 g) and glucose (20 g) per litre of distilled water, pH 6.0); and e) Yam Bean Medium (Pachyrhizus erosus L. Urban)- basic chemical composition (total protein (8.72 g), Starch (40.9 g) and glucose (11.14 g) per litre of distilled water, pH 7.0). Tuberous roots of yam bean were gently provided by Federal Rural University of Pernambuco (Northeast Brazil). The tuberous roots were washed with water and soap, for withdrawal of impurities. The Yam Bean Medium was prepared from tuberous roots peeled and rounded sliced (± 1,5 cm) and boiled in distilled water in the ratio of 1:2 (w/v) for 45 min (after initiate the boil). The broth was cooled, filtered in filter of paper and autoclaved at 121,5ºC, 15 min.

The sporangioles of C. elegans were harvested from cultures grown for seven days at 28ºC on Petri dishes containing PDA medium. A suspension of 10⁸ sporangioles/mL was used for inoculum. For fungal submerse cultivation, 10 mL sporangioles suspension were inoculated in Erlenmeyers flasks of 1000mL of capacity containing 290 mL of culture media. The flasks were incubated at 28ºC, in orbital shaker of 150 rpm, during 96 hrs. The mycelia were harvested, washed twice in deionizer water using a silkscreen nylon membrane (120 F), and submitted to lyophilization process. During C. elegans submerse cultivation in yam bean medium; aliquots were collected every 24 hrs for biomass, pH, glucose and total nitrogen were evaluated.

The glucose consumption was determined by the enzymatic colorimetric method (Labtest® Kit - Glucose oxidase), protein was utilized for nitrogen consumption determination, and the changes of pH were measured.

The process of extraction involved deproteination with 2% w/v sodium hydroxide solution (30:1 v/w, 90ºC, 2 hrs), separation of alkali-insoluble fraction (AIF) by centrifugation (4000 x g, 15 min), extraction of chitosan by Aceti acid under reflux (10% v/v acetic acid 40:1 v/w, 60ºC, 6 hrs), separation of crude chitin by centrifugation (4000 x g, 15 min) and precipitation of chitosan from the extract at pH 9.0, adjusted with a 4 M NaOH solution. Crude chitin and chitosan were washed on a coarse sintered-glass funnel with distilled water, ethanol and acetone and air-dried at 20ºC (Franco et al. 2004). The degree of deacetilation for microbial chitin and chitosan were determined by infrared spectroscopy (Roberts, 1992).

Sample of fungal chitin and chitosan (2 mg) were thoroughly blended with 100 mg of KBr, to produce 0.5 mm thick disks. The disks were dried for 24 hrs at 110ºC under reduced pressure. Infrared spectrometer was recorded with a Bruker 66 Spectrometer, using a 100 mg KBr disks for reference. Maximum absorption bands were determined.

The molecular weights of chitin and chitosan were determined by viscosity (Dos Santos et al. 2003). The viscosity of chitosan was determined.

The data were analyzed for significance using the Student’s t-test and chi-square test using STATISTICA program version 6.0 of Statsolt Inc., USA. All experiments were carried out in triplicate and the results are expressed as mean ± S.D.

The profile of growth of C. elegans (UCP 542) and chitin /chitosan production using yam bean medium and four different culture media are determined. A higher production of biomass, statistically significant (p = 0.0028) can be verified in the yam bean medium, with average dry weight corresponding to 20.4 g/L (Figure 1a). The best yield of chitin and chitosan from C. elegans (UCP 542) are obtained using yam bean medium, followed by  Sabouraud sucrose medium. The results are superior to the reported by Andrade et al. (2000) and Franco et al. (2005), which proposed C. elegans as a promising chitin source. C. elegans (UCP 542) grown in Hesseltine and Anderson medium shows a low biomass yield. The results are in agreement with the growth curve of C. elegans (IFM 46109) established by Andrade et al. (2000), Franco et al. (2005). The growth of C. elegans (UCP 542) in malt medium during 96 hrs, shows low average biomass, and similar to the reported by Synowiecki and Al-Khatteb (2003) using Mucor rouxii.

Chitin and chitosan production by C. elegans grown in yam bean medium are studied on the cultivation profile. C. elegans (UCP 542) growth curve for biomass, pH, nitrogen content and glucose consumption was analyzed. Biomass production increase rapidly up to 48 hrs, reached about the maximum of 24.3 g/L of dry weight. The result is superior to the value 10.41 g/L and 11.6 g/L reported by Andrade et al. (2000) and Franco et al. (2004), respectively, for C. elegans (URM 46109) grown during 96 hrs in Mucorales medium. The results showed glucose and nitrogen residual similar to the data reported by Andrade et al. (2000) and Franco et al. (2004). Amorim et al. (2001) suggesting that the remaining glucose and nitrogen as due to nitrogenous compounds like secondary metabolites present at the end of growth of the fungi metabolism and excess of carbon source in the medium.

The pH of yam bean medium oscillates between 7 and 5, during the culture time. The values of pH decrease during the exponential phase, due to the high glucose and starch concentration in yam bean medium. Franco et al. (2004) described constant values of pH during the “lag” phase and pH decrease during the exponential phase. Information of higher amount of the yam bean glucose and starch was previously mentioned by Sarangbin and Watanapokasin (1999), during the citric acid production. Amorim et al. (2001) reported that during growth of C. elegans the pH of the media drops in the first 24 hrs and remained low (between 3 and 4), during the first 96 hrs of cultivation, probably because of the interaction between the medium substrate and the release of ions from the cell.

Bests yields of chitin and chitosan yield extracted, to each 24 hrs, from C. elegans (UCP 542) grown in yam beam medium during 96 hrs was observed and best yields of the polysaccharides are obtained with 48 hrs of culture for chitosan and with 72 hrs for chitin. Tan et al. (1996), studying different Zygomycetes strains obtained similar results and observed that C. echinulata was the best chitosan producing strain. Although chitin production increase up to 72 hrs of culture, and decrease at 96 hrs. The higher chitosan yields in 48 hrs of growth suggest that during initial growth chitin is less crystalline and thus more susceptible to chitin deacetylase, and the chitosan formed prevails in acid pH. According to Amorim et al. (2005) optimum pH for chitin deacetylase activity is pH 4.5. During C. elegans growth the pH of the yam bean medium drops in the first 48 hrs, stowing high metabolic interchange between the medium substrate uptake and the release of ions from the cells. Amorim et al. (2001) reported higher yields of chitosan were found within 24 hrs of cultivation of M. racemosus and C. elegans at pH 3.5, which seems to be also a stimulating agent for production of this biopolymer. The data in the present work are in agreement with Pochanavanich and Suntornsuk (2002) and Nadarajah et al. (2001). Chung et al. (2004) demonstrated that chitin and chitosan content in the cellular wall of fungi change according to the species and these polymers usually show higher values in Zygomycetes.

The characterization of chitin and chitosan obtained from C. elegans in yam bean medium by infrared spectra are similar to those reported in the literature (Amorim et al. 2001; Andrade et al. 2000; Franco et al. 2005). The most significant parts of chitin and chitosan spectra are those showing the amide bands at approximately 1665, 1555 e 1313 cm^-1, which could be assigned to the C = O stretching, the N- H deformation in the CONH plane and the CN bond stretching plus CH₂ wagging. In a similar way, chitin from C. elegans shows bands in the amide II region, which were 1153, 1378 and 1558 cm^-1. The results are in agreement with Shigemasa et al. (1996), Andrade et al. (2003) and Franco et al. (2005). According to Dos Santos et al. (2003) the deacetylation and the regeneration process, cause disturbance in the initial crystalline reticulum of chitin, inducing a reordering of the hydrogen linking of chitosan. The data are in accordance with the reported in literature when comparing both chitin and chitosan infrared spectra obtained by microbiological methods (Andrade et al. 2000; Amorim et al. 2001; Pochanavanich and Suntornsuk, 2002; Dos Santos et al. 2003; Franco et al. 2005). Deacetylation degree (%DD) is an important parameter associated with the physical-chemical properties of chitosan (Pochanavanich and Suntornsuk, 2002). In the present study chitin and chitosan from C. elegans grown in yam bean medium present 6,2% DD and 85% DD, respectively. Amorim et al. (2001), Pochanavanich and Suntornsuk (2002) and Franco et al. (2004), reported deacetylation degree of chitosan from fungi between 80 to 90% DD. The average viscosimetric molecular weight (MV) of chitin and chitosan from C. elegans are in agreement with Nadarajah et al. (2001), Pochanavanich and Suntornsuk (2002) and Dos Santos et al. (2003).

The results present here described the high biotechnological potential of yam bean as an economic medium to chitin and chitosan production by C. elegans, and may be used to reduce the cost price of these polysaccharides production.

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