Carbohydrate polymers

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Carbohydrate polymers

Introduction

antibacterial chitooligosaccharide polymer carbohydrate

Chitosan is derived from chitin by deacetylation in the presence of alkali which is a copolymer consisting of (3 - (1 - 4) - 2-acetamido-D-glucose and - 4) - 2-amino-D-glucose units with the latter usually exceeding 80% (Arva-nitoyannis, Nakayama & Aiba, 1998).

Recent studies on chitin and chitosan have attracted interest for converting them to oligosaccharides, because the oligosaccharides are not only water-soluble but also possess versatile functional properties such as antitumor activity (Suzuki, Mikami, Okawa, Tokoro, Suzuki & Suzuki, 1986; Suzuki, Matsumoto, Tsukada, Aizawa & Suzuki, 1989; Tsukada et al., 1990), immuno-enhancing effects (Suzuki, 1996; Suzuki, Watanabe, Mikami, Matsumo & Suzuki, 1992), enhancement of protective effects against infection with some pathogens in mice (Tokoro, Kobayashi, Tayekawa, Suzuki & Suzuki, 1989; Yamada, Shibuya, Kodama & Akatsuks, 1993), antifungal activity (Hirano & Nagao, 1989; Kendra, Christian & Hadwiger, 1989), and antimicrobial activity (Hirano & Nagao, 1989; Uchida, Izume & Ohtakara, 1989). With respect to antibacterial activity, chitosan is superior to chitin since chitosan possesses a lot of polycationic amines which interact with the negatively charged residues of macromolecules at the cell surface of bacteria (Young & Kauss, 1983) and subsequently inhibit the growth of bacteria. The antibacterial effect of chitooligosaccharides has been shown to be greatly dependent on their degree of polymerization (DP) or molecular weight and requires glucosamine polymers with DP 6 or greater (Kendra & Hadwiger, 1984). In addition, the water-soluble chitooligosaccharides may be advantageous as antibacterial agents in in vivo system compared to water-insoluble chitosan.

We have previously reported (Jeon & Kim, 1999) that we could prepare chitooligosaccharides from chitosan by using an UF membrane in conjunction with enzymatic reactor to give chitooligosaccharides with three different molecular weight range such as a high molecular weight fraction (HMWCOS), a medium molecular weight fraction (MMWCOS), and a lower molecular weight fraction (LMWCOS). In the present study, the antimicrobial activity of these three fractions were examined against four Gram-negative, five Gram-positive and four lactic acid bacteria, to investigate the effect of molecular weight on the growth of microorganisms.

1. Materials and methods

1.1 Materials

Chitosan (degree of deacetylation: 89%, average molecular weight: 685,000) was donated by Kitto Life Co. (Korea). Chitosanase (694 units (U)/g protein) for the preparation of chitooligosaccharides was from Bacillus pumilus BN-262 and purchased from Wako Chemical Industries, Ltd (Japan). UF membrane reactor system for production of chitooligosaccharides was from Millipore Co. (USA). The microorganisms tested for antimicrobial activity were from Korean Collection of Type Cultures (KCTC) and American Type Culture Collection (ATCC).

1.2 Preparation of chitooligosaccharides using an UF membrane bioreactor

Chitooligosaccharides were prepared by continuous hydrolysis of chitosan in an UF membrane reactor system connected to an immobilized enzyme column reactor in which chitosanase from Bacillus sp. was adsorbed on chitin as a carrier for immobilization, according to our previous method (Jeon & Kim, 1999). The three different UF membranes used in the system had molecular weight cut offs (MCWO) of 10, 5, and 1 kDa. One percent chitosan solution (pH 5.5) was passed through the packed column reactor containing the immobilized enzyme at an output flow rate of 5 ml/minto obtain partially hydrolyzed chitosan (PHC) solution. PHC was continuously added to the UF membrane reactor system for the enzymatic conversion of PHC to chitooligosaccharides. Three kinds ofchitooligosac-charides (COS) prepared in the system were HMWCOS, these oligosaccharides passed through the 10 kDa MWCO membrane but not the 5 kDa membrane; MMWCOS, these passed through 5 kDa membrane but not the 1 kDa membrane and LMWCOS, these passed through the 1 kDa membrane. The yield and total reducing sugar content ofthe three oligosaccharides fractionated according to molecular weight were calculated by our previous method (Jeon, Park, Byun, Song & Kim, 1998).

1.3 Assays for antimicrobial activity

Antimicrobial activity of chitosan and three oligosacchar-ides was examined against various bacteria including four Gram-negative bacteria (Esherichia coli KCTC 1682, Escherichia coli O-157 ATCC 11775, Salmonella typhi KCTC 2424, Pseudomonas aeruginosa KCTC 1750), five Gram-positive bacteria (Streptococcus mutans KCTC 3065, Micrococcus luteus KCTC 10240, Staphylococcus aureus ATCC 6538P, Staphylococcus epidermidis KCTC 1917, Bacillus subtilis KCTC 1028), four lactic acid bacteria (Lactobacillus bulgaricus KCTC 3188, Lactobacillus casei KCTC 3189, Lactobacillus fermentum KCTC 3112, Streptococcus faecalis ATCC 10541). The assays were carried out by colony count on incubated agar plates. The mixture of 0.5 ml of the cultured bacteria, 0.5 ml of the autoclaved sample solution and 4 ml of 0.05 M acetate buffer (pH 6.0) was incubated with shaking at 37 °C for 1 h. In control samples, 4.5 ml of the acetate buffer was used. The mixture solution (1 ml) was diluted by 10-fold, added to Tryptic soy agar (TSA, Difuco) medium, plated on a plastic petri-dish, and then incubated at 37 °C for 24 h. After incubation, the colonies were counted to indicate bactericidal activity which was calculated by the following equation: Bactericidal activity (%) = [(C - T)/C] X 100, where C is the colony numbers counted on the control and T is those on the sample plate tested.

Minimum inhibitory concentration (MIC) was tested by two-fold serial broth dilution as follows. Bacteria culture (10⁵-10⁶ colony/ml) grown in 5 ml Tryptic soy broth (TSB) which contained 1 ml of the test sample was incubated at 37 °C for 18 h. MIC was defined as the lowest concentration of the tested sample at which the cell growth was not visible with naked eye or microscopy.

The inhibitory effects of chitosan and the oligosacchar-ides on the growth of E. coli were examined periodically by measuring the turbidity of the cultured medium at 640 nm. Either 5 ml of 0.05 M acetate buffer (pH 6.0) or 1% sample solution (pH 6.0) was added to the mixture of the cultured bacteria (0.5 ml) and TSB medium (44.5 ml) to give a final concentration of 0.1% and incubated at 37 °C.

2. Results and discussion

Chitosan as well as its hydrolysates has shown antibacterial activity and the inhibitory levels have been shown to be significantly dependent on the DP (degree of polymerization) molecular weight (Hirano & Nagao, 1989; Kendra et al., 1989; Uchida et al., 1989; Ueno, Yamaguchi, Sakairi, Nishi & Tokura, 1997). We have successfully obtained three fractions of chitooligosaccharides, based on their molecular weights, using the UF membrane reactor in our previous study (Jeon & Kim, 1999) and their antibacterial activity was examined in this study. As indicated in Table 1, the yields obtained by weighing the dried products of HMWCOS and MMWCOS were 12.6 and 9.4%, respectively, while that of LMWCOS was 78.0%. Total reducing sugar, which provides information on the hydrolytic level of the oligosaccharides was determined. The content of LMWCOS (229 mg/g chitosan) was greatly higher than others (51 and 74 mg/g chitosan for HMWCOS and MMWCOS, respectively). From the results of the previous study (Jeon & Kim, 1999), the molecular weight distribution of HMWCOS and MMWCOS ranged from 24 to 7 kDa and 6 to 1.5 kDa, respectively. The profile of LMWCOS consisted of oligosaccharides with DP in the range from pentamer to heptamer. These results revealed that the enzymatic hydrolysis of chitosan using the UF membrane bioreactor was suitable for the production of chito-oligosaccharides.

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