The crude shrimp head and skin materials have only a low economical value and are treated as biowaste or sold to animal feed manufacturers [1]. The shrimp biowaste in the tropical region contains 10-20 % calcium, 30-65% protein content and 8- 10% chitin on a dry basis [2]. Chitosan, which is obtained by N- deacetylation of chitin, has attracted attention due to its unique cationic character. Typically, chitosan is produced from waste generated from crustacean processing (e.g. shrimp and crab). Chitin and its derivative chitosan are of commercial interest due to their excellent biocompatibility, biodegradability, non-toxicity, chelating and adsorption power. With these characteristics, especially chitosan has many attractive applications in biotechnology, food and pharmaceutical industry, in cosmetics, environmental engineering, in agriculture and aquaculture. Though chemical procedures are widely used to produce chitin and chitosan utilizing shrimp waste as raw material [3], there is not much information how to improve the production process so as to produce chitosan with consistent quality, especially from stored material sources. The purpose of this study is to include partial autolysis in the existing protocol in order to improve the quality of chitosan product. This paper describes experiments to determine a suitable process for chitin/chitosan production from partially autolysed shrimp shell and suggests utilizing shrimp shells that have softened and partially degraded under carefully defined conditions.

Shrimp shells from black tiger shrimp (Penaeus monodon) were provided by a local shrimp processing factory. The shrimp shells were collected in big plastic bags with addition of ice to preserve the shrimp shell from decomposition. During transportation to the laboratory (transport time was only one and a half hour), the shrimp waste was kept cool and in the dark to avoid effects of direct sunlight. After arrival, the shrimp waste was directly used to produce chitosan as specified below. In this paper shrimp shell without further treatment is named fresh shrimp shell, shrimp shell stored in the freezer (at minus 18°C) for several months is named frozen shrimp shell.

Various procedures have been adopted to obtain high quality chitin by removing protein, inorganic material (mainly CaCO3) and pigments and lipid. Demineralization is usually carried out by treatment with HCl and deproteination by treatment with NaOH but other researchers have used other methods as well. The sequence of these processes has been varied, although in most chitin producing industries deproteination is carried out prior to demineralization [4]. In this research a modified method has been used in which shrimp shell was allowed to undergo partial autolysis before it was used to produce chitin and chitosan. The selected autolysis times were 1, 2, 3 and 4 days. Fresh shrimp shell used as the control for this experiment is marked as 0 day treatment (Fig. 1). The autolyzed shrimp shell was demineralized with 0.68 M HCl solution (1:5 w/v) at ambient temperature (28-32°C) for 12 h. The residue was washed and soaked in tap water for 6-8 h. It was then dewatered and deproteinized with 0.62 M NaOH solution (1:5 w/v) at ambient temperature for 20 h.

Fig. (1) A comparison between (a) the common process and (b) revised process under this study for the production of chitin and chitosan.

The chitin obtained from the revised process above was deacetylated in 12.5 M NaOH (1:5 w/v) solution at 65°C for 20 h. After deacetylation, the chitosan was washed and dried in sunlight and assayed for moisture content, ash content, protein content, degree of deacetylation, viscosity, solubility, turbidity and molecular weight [5]. The protein content in the chitosan sample was determined using the micro-biuret method [6]. The degree of deacetylation was analyzed by first derivative ultraviolet (UV) spectrophotometry [7]. Weight average molecular weight was determined by gel permeation chromatography (Waters GPC) with a differential refractometer detector. Dextrans of various molecular weights ranging from 9.9 x 10³ to 2 x 10⁶ Kd were used as standards. Ash content was determined by the standard AOAC method [8]. Various physico-chemical criteria were investigated under standard conditions, for chitosan in a 1% solution in 0.35 M acetic acid. Turbidity was assessed using a turbidity meter (Model 2100P portable turbidity meter, HACH Company, USA) and viscosity by a Brookfield Model DV - VII + Viscometer. Solubility was measured using the transglucosidase method [9]: the pH of 50 ml 1% (w/v) chitosan solution was adjusted to 4.8 with 30% (w/v) sodium acetate. Transglucosidase L-500 (Genencor International, 500 µl) was added. After incubation at 60°C for 24 h, the insoluble material was collected by filtration using a pre-weighed Whatmann GF/C filter paper (1.2 µm). The filter paper was dried and weighed and the amount of insoluble material was calculated from its weight gain.

The viscosity of a chitosan sample is a crucial parameter that indicates the quality of the chitosan sample. From the results obtained through this study, it can be concluded that chitosan samples with an enhanced viscosity can be obtained when the shrimp biowaste is allowed to a limited decay. In particular, the viscosity of chitosan samples extracted from fresh shells and treated for 1 day and from frozen shells treated for 2 days show a remarkable higher viscosity.

Normally, viscosity, solubility and molecular weight of chitosan are closely related. But the result of this study shows that in spite of the increase of the viscosity, other characteristics of chitosan such as the degree of deacetylation, molecular weight, solubility and turbidity have not changed compared to the chitosan from fresh material.