| Recently, enzymatic technology has become the ideal method for chitooligosaccharides preparation. However, it was commonly reported that wider ranges (<2 kDa) were obtained, which roughly limited the development of chitooligosaccharides with specific degree of polymerization (DP) for industrialization. The preparation of chitooligosaccharides in strict DPs with high yield but free from excessive cost by means of enzymatic and membrane separation technologies, which greatly promote the application of chitooligosaccharides in food and biomedicine industry.Thus, the approach combined with hybrid enzymes and membrane separation was carried out. Meanwhile, the enzymatic and separation mechanisms were investigated. Six commercial non-specific enzymes (chitosanase, cellulase, papain, xylanase, a-amylase, β-glycosidase and lysozyme) were used to compare the chitosanolytic activities (the pH and temperature have been optimized by the preliminary test). The results showed that chitosan could be well hydrolyzed by cellulase and papain when E/S was 0.5% (w/w). After 4 h, the viscosity-average molecular weight (Mv) of hydrolysis products was decreased to 24 and 32 kDa, respectively. The selected enzymes were combined with chitosanase, and the data demonstrated that the hydrolysis was accelerated by cooperation of chitosanase and cellulase, whereas conversely inhibited by papain. Thus, the optimum conditions of DP 6-8 chitooligosaccharides were determined as:2% Chitosan was dissolved in 0.4% (w/w) acetic acid with continuous stirring and pH was adjusted to 5.3 kept at 45℃.0.3% (w/w, based on chitosan) chitosanase and 0.8% cellulase were simultaneously added, and HPLC analysis illustrated that the yield of DP 6-8 chitooligosaccharides was up to 79.8% after 6 h.The hydrolysis products were purified by ultrafiltration (UF), and the influence of trans membrane pressure (TMP) and temperature on permeate flux and rejections of total proteins and DP 6-8 chitooligosaccharides were carried out. The results suggested that permeate flux increased with TMP or temperature increased. However, the concentration polarization would be accelerated if TMP was overhigh. Considering about the structural properties of membrane used,6.0 Bar and 50℃ were regarded as the suitable conditions during UF separation process. According to the conditions mentioned, the membrane was heavily polluted after 8 h, and it should be cleaned immediately. Six groups of abluents were selected to revive the polluted UF membrane. It was found that the permeate flux was totally recovered by adding the complex consist of 0.01 mol/L NaOH and 0.05% (w/v) SDBS.DP 6-8 chitooligosaccharides were further extracted by nanofiltration (NF). The effects of TMP and temperature on permeate flux and rejections of target products were investigated, and it was demonstrated that 16.0 Bar and 40℃ were described as the optimized parameters. At the same time, the rejections of glucosamine and DP 2-3 chitooligosaccharides were greatly affected by pH. HPLC profiles witnessed that over 90% of the three solutes could be successfully passed through membrane pores when pH was lower than 5.0, whereas 86.4% and 100% of dimer and trimer were rejected after pH reached to 9.0, respectively. After the NF concentrate was dried, the matrix-assisted laser desorption/ionization time-of-flight mass spectrum and HPLC profiles identified the concentrations of hexamer, heptamer and octamer in purified products. It demonstrated that the yield and purity of DP 6-8 chitooligosaccharides were up to 73.9% and 82.2% (w/w), which are satisfied with industrialization requirements. At last, a series of abluents were selected to compare the cleaning ability and the complex consist of 0.01 mol/L NaOH and 0.05% (w/v) EDTA-2Na was found to be most powerful among them. |
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