HONEYBEE (Apis mellifera) CHITOSAN: PURIFICATION, HETEROGENEITY AND HEMOCOAGULATING ACTIVITY

The aim of the research was to elaborate the method of chitosan preparation obtaining from honeybee corpses. It included the following stages: 1) washing of bee corpses with hot water; 2) delipidation of powdered material with petrol ether; 3) decalcification by EDTA at pH 4; 4) deproteination by 3 fold treatment with 5% NaOH 1 h at 70 oC; 5) bleaching of chitin with sodium hypochlorite; 6) deacetylation of chitin in 40% NaOH solution at 115 oC for 3 h; 7) purification of chitosan by its dissolving in 3% acetic acid and precipitation with ammonia at pH 8,5; 8) separation into three fractions precipitated at pH 6.4, 7.0 and 8.6. The yield of chitosan from dry bee powder was 8.5–10.0. A distinct diversity in the molecular mass of different chitosan fractions was revealed in the range of 80–320 kDa. Heterogeneity of chitosan samples was studied using gel permeation chromatography on Acrylex P-150 and electrophoresis in a slab of polyacrylamide gel with a stepwise gradient of acrylamide concentration 5, 10, 15, 20% in pH 4.5 buffer system. High molecular mass chitosan possessed blood coagulating activity, while low molecular mass fractions were poorly active. The rate of blood clot formation induced by active honeybee chitosan was 3 times lower comparing with that of chitosans obtained from crabs or shrimps.