CS200778B1 - Method of producing pullulan - Google Patents

Description

(54) Method of pullullan production

The invention relates to a process for the production of pullullan by culturing the microorganism Aureobasidium pullulans on suitable soils containing β (1-4) glucans.

Pullulan is an extracellular polysaccharide produced by some deuteromycetes of the species Aureobasidium pullulans. The structure is predominantly β-glucan with maltotriose units linked to each other by (, (1-6) glycosidic backbones) Pullulanes produced by different Aureobasidium pullulans strains differ in their detailed backbone structure, as well as in length and alignment.

So far, Pullulan is prepared from Aureobasidium pullulBns production medium containing a high, up to 10% D-glucose or sucrose concentration, with the actual pullullan formation only beginning when Aureobasidium pullulans is in the late adaptation phase and does not stop at the late adaptation stage. stationary phase. The extent of pullullan production depends on the type of strain used. While some strains of Aureobasidium pullulans do not produce this polysaccharide in fiber, some pullullan production strains may achieve up to 40% conversion of glucose or sucrose from production medium to pullullan. A drawback of the preparation of pullullan by most of the Aureobasidium pullulans is that the production of pullullan is accompanied by the formation of brown to black pigments that adsorb to the pullullan structure and make it practically impossible to prepare purified pullullan.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of pullullan by Aureobasidium pullulans

200 778

CCY 27-1-37, which aa is cultured on pCde with 2.5 to 10% D-glucose or sucrose or starch or / 5 (1 → 4) glucans or xylanes as carbon source for 10 to 15 days at 20 ° C to 30 ° C, whereby pullulan aa precipitates from the concentrated soil after removal of the biomass in 50-80% aqueous ethanol solution in a yield of up to 50% based on the carbon source in the additive.

The advantage of this process is the high yield of pullullan and the fact that the Aureobasidium pullulans CCY 27-1-37 strain produces pullulene free of pigments and other accompanying substances for the high carbon source addition. The pullulan produced by said strain has a typical structure of regular recurring maltotriose units, coupled to each other with (1-6) glycosidic bonds, and only about 3% of the glucosyl units are bound in the side chain of α (1-e-4) binding. The average molecular weight of the pullullan produced by this strain is 238,000. Further purification of the pullullan can be accomplished by precipitation from an aqueous ethanol solution or by column chromatography.

When preparing pullullan with Aureobasidium pullulans CCY 27-1-37 by cultivation on polysaccharide or agricultural waste sub-substrates, appropriate enzyme equipment of this strain is utilized with cellulytic, amylolytic and xylolytic enzyme activity. Obtained pullullan with the structure of regular repeating maltotriose units bound to each other by glycosidic bonds, obtaining in a pure state is suitable for use in the pharmaceutical industry (sedative, antidote against Salmonella sp. Toxins, protection of animal tissue against radioactive radiation, and for the preparation of maltotriosis by the cleavage of pullullan by the enzyme pullulanase from Klebsiella aerogenee.

Example 1

Aureobasidium pullulans CCY 27-1-37 is grown in a fall (0.5 L) containing 10% sucrose, 0.5% K ₂ HPO ₄ , 0.1% NaCl, 0.02% MgSO ₄ , 0.06% ( NH ₄ ) ₂ SO ₄ and 0.04% yeast autolysate, the pH being. adjusted to 7.2 on a reciprocating shaker at 25 ° C for 20 days. After that time, the Aureobasidium pullulans biomass is filtered off and concentrated in a vacuum evaporator to 1/20 of the feed volume and precipitated with twice the volume of ethanol. The precipitate was allowed to stand at 2 ° C for 2 hours, then centrifuged (5 minutes, 2000 g), dissolved in water and dialyzed. A white polysaccharide of up to 25 g dry weight is obtained. a more violent purification of pullullan from the accompanying proteins (about 2% by weight of pullullan) can be accomplished by dissolving pullullan in 0.05 N NaOH and, after stirring for 3 hours, repeated precipitation with 2 times ethanol and dialysis against water. A greater purification step was achieved on a Sepharose 6B column (25 x 1.5 cm) eluting with 0.1 U Tris-HCl + 0.1 M NeCl pH 7.0. Purified pullullan is dialyzed against distilled water and lyophilized.

Example 2

As in Example 1, the difference is that, instead of sucrose, 10% hydroxyethylcellulose is used as the carbon source in the production additive. Treatment of pullulan from solution after 12-day cultivation similar to that described in Example 1.

Example 3

As in Example 1, except that beech wood is used as a carbon source (instead of saccharose). Treatment of pullullan from solution after 10-day cultivation, similar to Example 1.

Example 4

As in Example 1, with the difference that instead of sucrose a, scrap is used as a carbon source (7% by volume of culture medium). The pullulanate released into the solution of culturing is obtained after removal of the biomass and scrap residues by filtration similar to that described in Example 1.

The invention has utility in the preparation of pullullan polysaccharide for the needs of human medicine as well as for the food industry.

Claims (1)

Beechwood β-xylan xylan. Treatment of pullul from solution after a 10-day culture, similar to that described in Example 1. Example 4 As shown in Example 1, except that instead of sucrose and a carbon source is used (7% per volume of cultivation) . Pullulan released into the solution of the culture is obtained after removal of the biomass and tails by filtration similar to that described in Example 1. The invention is of use in the preparation of pullul polysaccharide for human medicine and the needs of the food industry. SUMMARY OF THE INVENTION A method of producing pullulan, characterized in that Aureobaeidium pullulans CCY 27-1-37 is cultured in the pole with 2.5 to 10% - D-glucose or sucrose or starch or / (5 (1-4) glucans or xylans as a carbon source for 10 to 15 days, at 25 ° C, while the pullulan is precipitated from the concentrated stage after removal of the biomass in a 50-80% aqueous solution of ethanol.