The Studies On Fermentation And Modification Of Pullulan

In this paper, the medium and cultivation conditions of pullulan production were optimized. The technology of pullulan production was investigated. The production of maltotriose by hydrolysing of pullulan with pullulanase was studied. Water absorption resins of pullulan graft methyl acrylate (PU-g-MA) using ceric ammonium nitrate (CAN) as an initiator had been investigated under nitrogen atmosphere in aqueous medium.A strain of Aureobasidium pullulans, AP329, was used for the production of pullulan by employing hydrolysed sweet potato starch (HSPS) as a carbon source. Hydrolysates produced by a-amylase alone gave the lowest yields of pullulan and biomass. In contrast, continuous hydrolysis with P-amylase and pullulanase gave higher yields, but prolonged hydrolysis with amyloglucosidase did not improve the yield further. The maximum pullulan yield and biomass were achieved at the dextrose equivalent value (DE) of 48. The optimum cultivation conditions were as follows:pH, 5.5; fermentation time,4d. The HPLC analysis of polysaccharide revealed that the Mw of pullulan was 420000 Da and nearly 100%more than that of sucrose. The optimum cultivation medium contained (g/L), HSPS,50.0; yeast extract,2.0; KH₂PO₄, 5.0; (NH₄)₂SO₄,0.6; KCl,0.5; MgSO₄·7H₂O,0.2; NaCl,1.0, distilled water 1 L and pH,5.5. The production of pullulan increased more than 50% by employing this new cultivation medium after 4 d fermentation. Results of this work indicate that it that HSPS is a promising carbon source for the production of pullulan economically.The effect of nitrogen source on the production of pullulan synthesized by A. pullulans AP329 was investigated. Pullulan production was affected by the nitrogen source in the medium, and its yield fell when excess nitrogen source was present. When NaNO₂ was the nitrogen source, maximum pullulan production was achieved at 0.3 g/L. With (NH₄)₂SO₄, pullulan yields reached maximum at 0.6 g/L. At the same time, biomass accumulation increased as the level of nitrogen source raised. Effect of initial pH of the medium on pullulan production was associated with the nitrogen source used. With NaNO2, the maximum production was observed at the initial pH of 6.5. In the case of (NH₄)₂SO₄, the maximum amount of pullulan in the broth was achieved at an initial pH of 5.5. The optimal cultivation medium contained (g/L), HSPS,50.0; yeast extract,2.0; KH2PO4,5.0; NaNO₂,0.3; KCl,0.5; MgSO₄·7H₂O, 0.2; NaCl,1.0, distilled water 1 L and pH,6.5. The production of pullulan increased more than 10% by using this new cultivation medium after 4 d fermentation. Results of this experiment indicate that NaN02 is a promising nitrogen source for production of pullulan.To obtain pure biopolymer from the fermentation broth, heat treating, cell harvesting, broth concentration, removal of the melanin pigments co-produced during fermentation, precipitation and drying were necessary. Heat treating at 80℃for 20 min could kill the cells and remove most of the protein in the fermentation broth and almost had no effect on the recovery of pullulan. Centrifugation of the fermentation broth at 10000 rpm for 15 min gave cell pellets that were discarded and a green-black supernatant containing melanin pigment. The supernatant was concentrated to 10% (w/v) under vacuum and demelanized by oxidation with 4%(v/v) H₂O₂at 60℃, pH 10 for 30 min, Precipitating with ethanol and drying at 90℃for 30 min. The resulting pure pullulan was comparable in color and texture to that of commercial samples.The technology for the production of maltotriose by hydrolysing of pullulan with pullulanase revealed that the optimum conditions were 1) temperature,45℃; 2) pH, 5.0; 3) time,6h; 4) pullulanase, 10ASPU/g and pullulan,3% (w/v). Higher or lower temperature, pH and the concentration of pullulan decreased the DE values of the hydrolysates. Prolonged reaction time and higher concentration of pullulanase could not increase the DE values of the hydrolysates. The powder of maltotriose was obtained by treating the hydrolysates with heating, filtrating, concentrating, precipitation and drying. The profile of HPLC showed that the maltotriose content of the product was more than 93.5%(w/w). The yield of maltotriose was 87.3% (w/w, pullulan) and 49.9% (w/w, sweet potato starch), respectively.Water absorption resins of pullulan graft methyl acrylate (PU-g-MA) using ceric ammonium nitrate (CAN) as an initiator had been investigated under nitrogen atmosphere in aqueous medium. The investigation showed that the optimal conditions for grafting were:[CAN]=0.004 mol/L which was added in 1 mol/L nitric acid; [MA] = 0.0465 mol/L; reaction time,180 min and temperature,40℃. The percentage grafting (%PG) was affected by higher concentration of initiator, monomer, and high temperature. Prolonged reaction time could not improve %PG further. The graft copolymer was analyzed by gravimetrical method and infrared spectroscopy. The water absorption capacity of the resins was related to %PG When %PG. was below 15.3% (w/w), the water absorption capacity of the resins increaseded significantly with the increase in %PG, and when %PG. was above 15.3% (w/w), the water absorption capacity of the resins decreased sharply with the increase in%PG...