DD261609A1 - PROCESS FOR PREPARING A POLYSAC CHARIDE - Google Patents

Abstract

The invention relates to a novel process for the production of a microbial polysaccharide of the pullulan type by fermentation using the microorganism Pullularia pullulans ZIMET 43826 and subsequent purification of the resulting crude product using sodium hypochlorite and activated carbon in aqueous-alcoholic solutions.

Description

Field of application of the invention

The invention relates to a process for the preparation of a microbial polysaccharide of the pullulan type in pure form, which is useful in the food industry as well as a biochemical.

Characteristic of the known state of the art

Microorganisms are able to synthesize a wide range of polysaccharides of different chemical structure, have found versatile applications in the food industry and technology or are potentially used for a variety of purposes (BAIRD et al., Biotechnology 1983, pp. 778-783; CATLEY, Progr. Ind. Microbiol., Vol., 18, 130-200 (1983)). Among these polysaccharides, pullulan-type glucans are of interest because they are of interest both in the food industry (biodegradable confectionery packaging, preparation of dietetic preparations) and can also be used as a biochemical substrate for the determination of activities of amylolytic enzymes, eg pullulanase (see eg RENNEBERGj et al., Biotechnol. Lett.7, 809-812 [1985]). Chemically, pullulan consists of linear polymers (MW> 20000), in which each maltotriose units are linked together as a basic structural element in 1.6 position (BENDER et al., Biochem. Biophys. Acta 36, 309-313 [1959]).

It is known that fungal microorganisms of the genus Pullularia, preferably of the species Pullularia pullulans (Aureobasidium pullulans) accumulate extracellular pullulan when cultivated on suitable carbohydrate-containing nutrient media (UEDA et al., Appl.Mikrobiol.11, 211-215 [1963]). The disadvantage is that only relatively small amounts of pullulan are produced, or that slime-forming and colored by-products are released into the medium and isolated together with the Rohpullulan that reduce the suitability of the product as a food additive or as a substrate for enzymatic reactions.

Object of the invention

The invention serves to prepare a pullulan type microbial polysaccharide in order to extend the range of known processes for the preparation of glucose-containing polymers by a new, effective process for producing pure end product.

Explanation of the essence of the invention

The invention has for its object to describe a technologically simple process by means of which a microbial polysaccharide (glucan) of the pullulan type can be produced by fermentation using cheap starting materials in good yields and can be obtained in pure form.

According to the invention, this object is achieved in that a microorganism of the genus Pullularia, preferably the species P.pullulans and in particular the strain P.pullulans Gr 3-66, under aerobic and sterile culture conditions in a liquid medium with known carbon and nitrogen sources and is cultivated with the usual in the fermentation industry mineral salt additives to the medium under submerged conditions.

The microorganism P.pullulans Gr3-66 has been deposited in the ZIMET Depository for Microorganisms, Central Institute for Microbiology and Experimental Therapy of the GDR Academy of Sciences, DDR-6900 Jena, Beutenbergstrasse 11, under the registration number ZIMET 43826.

Cultivation and maintenance of the microorganism of the genus Pullularia used, preferably of the species P.pullulans and in particular the new strain ZIMET 43826, is carried out as follows:

Lyophilized mycelial fragments are inoculated on nutrient agar, and subsequently in liquid, previously sterilized culture media, and the resulting mycelium is in a known manner at a temperature between 20 ° C and 38 ⁰ C, preferably 26 ° C, over a period of 2 days to 6 Days, preferably 5 days, cultured at an acidity that is consistently above pH 4.0. In the further embodiment of the method, an acidity course is selected which is at the beginning of the fermentation between pH 5.5 and pH 8.3 and during the fermentation by addition of alkali, preferably NaOH, in the range of pH 4.0 to pH 6 , 0 is held. As carbon and nitrogen fillers as well as mineral salts, the starting materials customary in the fermentation industry can be used. The fermentation of the strain used can be carried out in steep-breasted bottles and round-bottomed flasks of various contents, in glass fermenters or V2A tanks. The detection of the formation of the polysaccharides is carried out by measuring the increasing viscosity of the fermentation solution and the consumption of the substrate used.

The workup is initiated at the end of the fermentation by separation of the culture solution and separation of the biomass (filtration).

Crude polysaccharide is precipitated by the addition of hydrophilic organic solvents to the culture solution as a deep brown amorphous mass. The further purification is carried out by dissolution in hot water and subsequent alternate treatment with activated carbon and hypochlorite solution, preferably sodium hypochlorite solution, and reprecipitation from the aqueous solution with hydrophilic organic solvents such as methanol, ethanol or acetone, this cleaning and precipitation procedure optionally is repeated. It will contain colorless polysaccharide characterized by the following properties:

Appearance: white powder (after grinding)

Chemical analysis: C40.03% H6.05%

Specific rotation: [a] ₅₄₆ = + 166.6 ° (water)

[a] ° = + 118.9 Hydrolysis: 2NHCl, 98 ° C, 6min. 60min.

gives mixtures of maltotriose and glucose,

Total hydrolysis in 2N HCl (8h, 98 ° C) gives glucose

The advantages of the invention are seen as follows:

The process produces a pure polysaccharide comparable in properties to pullulan.

- The microbiological process using P.pullulans ZIMET 43826 as a microorganism leads to the recovery of crude product in comparatively very good yields.

- The thus obtained by its deep brown color and concomitant Schleimstoffanteile unusable crude product can be effectively cleaned by treatment with decolorizing agents such as hypochlorite and activated carbon and freed of impurities. This provides a product which is potentially useful for biochemical purposes and applications in the food industry.

Exemplary embodiment

In one embodiment, the invention will be explained in more detail.

Lyophilized mycelial fragments are streaked onto AL-53 agar medium and grown at 28 ° C for 10 days. Composition of the AL-53 medium: 3.0g sucrose 15.0g dextrin 0.1g urea 0.5g NaCl 0.5g KH ₂ PO ₄ 1.0g yeast extract 5.0g peptone

1.0ml FeSO ₄ (1% saline) 15.0g Thread agar 1000ml Aqua dest pH 7.2

Inoculum cultures are obtained by sowing mycelial pieces of an agar culture into a medium of the following composition (g / l):

Soy meal 40, glucose 40, NaCl 2.5, NaCO ₃ 5, KH ₂ PO ₄ 0.25, pH 6.3. The second preculture takes place on the same medium. After 48stündigerBebrütung the preculture (500ml reagent bottles, 80ml content, 25 ⁰ C, round oscillating table, 180 RPM), the stepwise scaling performed by transmission of culture samples in 51-reagent bottles (12:51) medium and a further 24 hours incubation, 27 ° C.

For the cultivation in fermenters, a medium of the following composition is used (g / l): glucose 60, peptone 5, (NH ₄ J ₂ SO ₄ 5, MgSO ₄ .7H ₂ O 5, CaCO ₃ 10, KH ₂ PO ₄ 0.5. Aeration is carried out with sterile air (1: 1), the fermentation time is 120 h, the stirring speed is 280 rpm Foaming can be controlled in a manner known per se by adding small amounts of silicone-containing antifoam agents pH 5.5-6.0 (NaOH).

The yield of crude polysaccharide (dry matter after ethanol precipitation) is 20-24 g / l after 120 hours of fermentation. After separation of the culture solution from the mycelium, it is added in the ratio 1: 1 with ethanol (or methanol), the polysaccharide is precipitated as a brown-black gelatinous mass and recovered by filtration.

The crude product is then dissolved in the same volume of hot water (20 L) and treated with 500 g of activated carbon. After filtration, the solution (151) is again heated to 90 ⁰ C and treated for 30 min with 1.51 sodium hypochlorite solution (3%). It is then filtered and precipitated from the filtrate, the polysaccharide with the same volume of methanol or acetone. The final cleaning is carried out by re-dissolution of the precipitated product in hot water and boiling with 200 g of activated carbon (0.5 h, 95 ⁰ C). After filtration, the polysaccharide is precipitated from the cooled solution by addition of methanol or acetone. The resulting colorless product is dried at 50 ^° C. to constant weight. Yield: 65g pure white polysaccharide.

Claims (6)

A process for the preparation of a pullulan-type polysaccharide by aerobic fermentation of a fungus of the genus Pullularia in sterile, liquid nutrient media containing a carbon and a nitrogen source and mineral salts, characterized in that The microorganism P.pullulans Gr3-66 (strain ZIMET43826) is cultured at an acidity constantly above pH 4.0, and - The polysaccharide formed is purified after precipitation from the culture solution by alternating treatment with activated carbon and hypochlorite solution. 2. The method according to claim 1, characterized in that the strain P.pullulans Gr3-66 for a period of 2 days to 6 days at a temperature between 20 ° C and 38 ° C is cultivated. Process according to claims 1 and 2, characterized in that strain P.pullulans Gr3-66 is cultured for a period of 5 days at a temperature of 26 ⁰ C and an acidity which is between pH 5.5 and pH 8.3 at the start of fermentation and subsequently maintained in the range of pH 4.0 and pH 6.0. 4. The method according to claim 3, characterized in that the acidity by addition of alkali, preferably NaOH, is maintained in the range between pH 4.0 and pH 6.0; 5. The method according to claim 1 to 4, characterized in that after separation and filtration of the culture solution by hydrophilic organic solvent, preferably by ethanol, methanol or acetone, a crude polysaccharide is precipitated. 6. The method according to claim 1 and 5, characterized in that the crude polysaccharide by dissolution in hot water and alternating treatment with activated carbon and sodium hypochlorite solution and reprecipitation with hydrophilic organic solvents, optionally with repetition of this cleaning and precipitation step, further to a polysaccharide from Type of pullulan is cleaned.