Chitosan Isolated from Horseshoe Crab Tachypleus Gigas from the Malay Peninsula

Introduction

Horseshoe crab, a chelicerate arthropod, is among the world’s oldest and most fascinating creatures. It has survived from the ancient time until now and thus, it is called as the living fossil. In Malaysia, there are three species which are commonly found in the coastal water; Tachypleus gigas, in the estuary area, Carcinoscopius rotundicauda, and the biggest horseshoe crab strictly found in Sabah water, Tachypleus tridentatus. The natural land barrier of the Malay Peninsula has contributed to the genetically differentiated population from those of the Indian Ocean and those of the South China Sea [1], at least for T. gigas. Horseshoe crab also has extraordinary defense mechanism due to its blood property. Nowadays, horseshoe crab has been recognized for its contribution in medicine and pharmaceutical science. Its carapace contains a compound called chitin and essentially, chitins as well as its derivative chitosan have played big roles in various fields of applications [2].

Chitin is the second most important and ubiquitous natural polysaccharide after cellulose and it can be extracted from the shell of horseshoe crabs, marine invertebrates, insects, fungi and yeast. It has a polymeric substance derived from crustaceous shell which is a composed of β(1à4)-linked 2-acetamido-2-deoxy-β-D-glucose. This polymeric substance is an acetamido group (-NHCOCH₃) instead of hydroxyl group at C-2. Chitin functions like cellulose as structural polysaccharide and its derivative chitosan has a linear polymer of α(1à4)-linked 2-amino-2-deoxy- β-D-glucopyranos [3].Chitosan is a fiber-like substance derived from chitin by N-deacetylation and comprised copolymer of N-acetylglucosamine and glucosamine. It is obtained from chitin by deacetylation; a process where acetyl group is removed. The degree of deacetylation determined the chitosan produced from chitin. The removal of enough acetyl group (CH₃-CO) from chitin has resulted the chitin molecule and its derivative, chitosan, to be soluble in most diluted acid. The acetyl content of the polymer has become the actual variation between chitin and chitosan.

Chitin and chitosan as biopolymer have many functions and utilizations. Nonetheless, there are still restrictions in the product properties due to certain processes [4]. The standard procedure of isolation involved of three basic steps; deproteinization (protein separation), demineralization (calcium carbonate and calcium phosphate separation) and deacetylation (removal of acetyl group) which is for the subsequent conversion of chitin to chitosan. The conversion of chitosan where some or all acetyl groups are removed from chitin is generally achieved by treatment with concentrated sodium hydroxide solution (40-50 %) at 100 ºC or higher temperature [5]. As reported before [6], physicochemical characteristics of chitosan affects its functional properties such as dye binding capacity, water binding capacity, fat binding capacity, and emulsifying capacity, which also differ depending on the crustacean species and its preparation methods.

Antimicrobial resistance is a threat to mankind because most of the infection causing bacteria has become multidrug resistant [7].The antibacterial activities of chitosan have been demonstrated against gram-positive and gram-negative bacteria by many researchers [8, 9, 10]. The polycationic nature of chitosan is prerequisite for antimicrobial activity. Eventually, the interaction between the positively charged NH3+ groups and the negatively charged microbial cell surface contribute to the leakage of protein and other intracellular components of the microbial cells, ultimately resulting in the impairment of vital bacteria activities [11, 12,13].

Considering the possibility of genetic difference due to different marine environment [1], physicochemical and biological properties of chitosan from those horseshoe crabs could also be different. Hence, the general objective for this research was to study the physicochemical properties and antibacterial activities of chitosan prepared from horseshoe crab carapace obtained from different coastal landing area of the Malay Peninsula. The sampling site was at Balok in the east coast and Muar in the west coast of the Malay Peninsula. Balok received sea water from the South China Sea, while Muar from the Straits of Malacca.

Methadology

Sample Collection

The amount of 40 live horseshoe crabs (Tacypleus gigas) from coastal area of Balok, Pahang (East coast) and coast area of Muar, Johor (West coast) were collected from those caught by fishermen. The horseshoe crabs were then brought to oceanography laboratory and kept in the freezer.

Isolation of Chitin and Chitosan

The carapaces were removed from the body of the horseshoe crabs. The loose tissue and the muscle on the carapaces were scraped free and washed thoroughly with tap water. Then, the carapaces were completely dried using MEMMERT oven at 60 ºC overnight. The dried carapaces were then weighed and recorded. The dried carapaces were cut into smaller size before grinding using SHARP commercial glass blender. The ground horseshoe crab carapaces were sieved using a 250micrometer sieve mesh and sieve shaker to get a uniform particle size. Then, the carapaces powder was weighed and kept in the Schott bottle before being extracted. Deproteinization was carried out [14], followed by demineralization, and then deacetylation [15].

Characterization of Chitosan from Horseshoe Crab Carapace

Chitosan samples were prepared in a form of thin disk, which was a mixture of KBr and chitosan’s powder with the ratio 3:1. The sample mixture was thoroughly mixed, ground into fine particles, and then pressed in order to form homogenous thin sample disc. Next, the disc was kept in a desiccator for 16 hours before placing the KBr disc in a sealed plate for scanning. The spectra of the chitosan discs were obtained by using FT-IR with the frequency range of 4000-400 cm^-1 [16]. The functional groups of the chitosan samples were confirmed by comparing the spectra obtained with the spectra obtained in the reference study [17].

The deacetylation degree (DD) of the chitosan samples were calculated using the computational equation baseline [18] as below:

A₁₆₅₅ refers to the absorbance at 1655 cm^-1 of the amide-I band as a measure of the N-acetyl group content, while A₃₄₅₀ refers to the absorbance at 3450 cm^-1 of the hydroxyl band as an internal standard to correct for disc thickness or differences in chitosan concentration powder form. The factor ‘1.33’ denoted the value of the ratio of A₁₆₅₅/ A₃₄₅₀ for fully N-acetylated chitosan.

The moisture content of horseshoe crab chitosan from Balok and Muar were determined using moisture analyzer (PRESICA XM 50). The ash content was calculated by using the formula below:

For solubility, 0.3 g of horseshoe crab chitosan was weighed and kept in a pre-weighted centrifuge tube in triplicates. The mixtures of chitosan and acetic acid were centrifuged (UNIVERSAL 320R, Zentrifugen) at 9,000 rpm for 15 minutes. The supernatant liquid was removed again and the residue was dried at 60 ºC for 24 hours [17]. Finally, the dried particles were weighed and the percentage of solubility was calculated with the given formula:

The 1% (w/v) of chitosan solutions was prepared in 1 % acetic acid to analyse its viscosity. Then, the viscosity was measured by using viscometer (VIBRO SV-10). The result appeared when it became constant and was recorded in mPa.s (1cP = 1 mPa.s).

Antibacterial Activity of Horseshoe Crab Chitosan by Disc Diffusion Method

The disc diffusion method was used to test the antibacterial activity of horseshoe crab chitosan from Balok and Muar. The bacteria strains used for this antimicrobial test were Staphylococcus aureus and Escherichia coli. Müeller-Hinton agar medium was poured into petri dishes and allowed to solidify. A total of 100 microLiter diluted inoculum of bacteria strains was spread evenly on the agar plates by using hockey stick spreader. The sterilized paper discs were impregnated with 100 microLiter of diluted chitosan solution (1 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1 % (w/v)) and placed on surface of the agar mediums with 1 % solution of acetic acid as negative control and streptomycin as positive control. Then the agar plates were incubated at 37 ºC for 24 hours.

Result and Discussion

Yield of Chitosan from Horseshoe Crab Carapace

The chitosan yield in this study ranged from 12.7-15.0 % depending on the source of horseshoe crab [19] and method used [20]. The yields of chitosan were comparable for both coastal areas (Table 1) but lower than the percentage yield reported before [14]. Excessive removal of acetyl group from the polymer during deacetylation could cause loss of sample weight or could be due to the loss of chitosan particles during filtering and washing [21]. On the other hand, chitosan yield might be depending on the length and alkaline and acid concentration used during deproteinization and demineralization [22].

Table 1: Percentage yield of chitosan isolated from horseshoe crab carapace obtained from Balok and Muar in the Malay Peninsula.

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