I have used following method for the colloidal chitin preparation. Ten grams of chitin was slowly dissolved in 400 ml of concentrated HCl with stirring at 4°C. The mixture was incubated in a water bath at 37°C until viscosity decreased. To this mixture 4.0
liters of sterile distilled water was added and left overnight at 4°C. The
supernatant was slowly decanted and the precipitate was collected on a filter
paper and washed extensively with sterile distilled water to attain neutral pH.
But still the prepared chitin is insoluble in water. I want to check the chitinase producing activity of bacterial culture, that's why i required it.. So please suggest if there is any other method.
This method i also followed in PhD
The colloidal chitin preparation was done according to the method described by Rodriguez et al., 1983. The colloidal chitin suspension was prepared by dissolving 200 g of ground Crustacean chitin (0-25 mm) in 1.8 liter of conc. HCl. The acid was added to the chitin with stirring and the mixture was allowed to stand at 20°C with intermittent stirring until dissolved (1.5-2 hr). As a polyacetal, chitin is sensitive to acids and can be easily hydrolyzed even more than cellulose, that is why caution must be taken during preparation of colloidal form during the time contact with HCl which must not be exceeded. The solution was poured with stirring into a 60L container half-filled with tap water. A suspension of chitin in water was formed and additional water was added to have the final volume of 50L. The suspension was allowed to stand overnight allowing the chitin to settle and form a concentrated suspension. The supernatant liquid was slowly siphoned off and tap water was added to the resuspend chitin. This process of washing with tap water was repeated 4 times, followed by 3 washes with demineralized water. Note that the washing step may take days until it is satisfying that most of the acidity is washed. After final wash, the suspension was passed through a triple layer of 0.1-mm-mesh nylon cloth to remove large particles. This was replaced by centrifugation at 2000 rpm under cooling using Beckman GS-6R table top centrifuge.
The resulting chitin was suspended and had a pH of 5.5-6.0 and was stored in the dark at 4°C until used, the chitin content of the suspension was determined gravimetrically after drying 10 ml-samples at 80°C for 24 h. The stock suspension was used to prepare others of lower concentration by diluting with demineralized water immediately prior to use. An alternative method was held on by vacuum drying of the freezed form at 35°C, as mentioned by Willis and Scott (1988), because according to its sensitivity to heat, even in the 40-50°C range it may denature with time. The drying process was done in an electric oven equipped with the vacuum facility under 650 mmHg over 3 days.
This method i also followed in PhD
The colloidal chitin preparation was done according to the method described by Rodriguez et al., 1983. The colloidal chitin suspension was prepared by dissolving 200 g of ground Crustacean chitin (0-25 mm) in 1.8 liter of conc. HCl. The acid was added to the chitin with stirring and the mixture was allowed to stand at 20°C with intermittent stirring until dissolved (1.5-2 hr). As a polyacetal, chitin is sensitive to acids and can be easily hydrolyzed even more than cellulose, that is why caution must be taken during preparation of colloidal form during the time contact with HCl which must not be exceeded. The solution was poured with stirring into a 60L container half-filled with tap water. A suspension of chitin in water was formed and additional water was added to have the final volume of 50L. The suspension was allowed to stand overnight allowing the chitin to settle and form a concentrated suspension. The supernatant liquid was slowly siphoned off and tap water was added to the resuspend chitin. This process of washing with tap water was repeated 4 times, followed by 3 washes with demineralized water. Note that the washing step may take days until it is satisfying that most of the acidity is washed. After final wash, the suspension was passed through a triple layer of 0.1-mm-mesh nylon cloth to remove large particles. This was replaced by centrifugation at 2000 rpm under cooling using Beckman GS-6R table top centrifuge.
The resulting chitin was suspended and had a pH of 5.5-6.0 and was stored in the dark at 4°C until used, the chitin content of the suspension was determined gravimetrically after drying 10 ml-samples at 80°C for 24 h. The stock suspension was used to prepare others of lower concentration by diluting with demineralized water immediately prior to use. An alternative method was held on by vacuum drying of the freezed form at 35°C, as mentioned by Willis and Scott (1988), because according to its sensitivity to heat, even in the 40-50°C range it may denature with time. The drying process was done in an electric oven equipped with the vacuum facility under 650 mmHg over 3 days.
i have in a paper that they used NaOH, after treating with HCl and distilled water in the manufacturing of colloidal chitin, can you please guide me why they did so?
The treatment of the colloidal suspension with NaOH after acid treatment is to ensure that the pH rose to around 4.5-5 rapidly. This is normally achieved by washing with water repeatedly. The NaOH will neutralize the excess acid present and bring about increase in the pH.
i have simplified method of preparing colloidal chitin used for screening if chitinase:
is dissolved in non aqueous solution include " LiCl (5%)/DMA or LiCl/N-methyl-2-pyrrolidone or CaCl2/MeOH, or hexafluoroisopropyl alcohol.
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