CZ20024026A3 - Fermentation process and isolation of extracellular glucan using Claviceps species fungi - Google Patents

Abstract

In the present invention, there is disclosed a fermentation process and isolation of extracellular glucan using Claviceps species fungi wherein the process is characterized in that Claviceps species fungi are used as a producer producing exopolysaccharides and which does not produce alkaloids and like biologically active substances, particularly from a group of Claviceps viridis and Claviceps maximensis species. Submersion cultivation is carried out at a temperature ranging from 18 to 28 degC, on a synthetic culture medium, biomass of the production microorganism is then removed after fermentation from the culture liquid by filtration or centrifugation and the retenate is stabilized after dialysis to a final appearance by lyophilization. A non-amorphous glucan form is prepared in such a manner that the retenate is exposed to the action of a direct current within an electrocoagulation device with an iron or aluminium sacrificed electrode at current density ranging from 0.05 to 0.8 mA/cme2 of surface of immersed electrodes at voltage gradient in the range of 0.2 to 2 V/cm of a distance between two electrodes, or in the electrocoagulation device, there can be used stainless steel electrodes and heavy metal salts are added to the solution, preferably aluminium, copper, iron, cadmium or lead salts in an amount ranging from 2 to 15 ml of the salt saturated solution per 1 liter of the culture liquid.

Description

The invention relates to a two-step process for the preparation of extracellular glucan based on the use of fungi of the genus Claviceps sp.

BACKGROUND OF THE INVENTION

1,3 / 1,6 β-glucan (hereafter glucan) is a polysaccharide whose exceptional properties have been studied in the context of iunoprotective action (US Patent 5,576,015 and 5,702,719) (Hiroiku Ueno Beta-1,3-DGlucan, its Immune Effect and its Clinical Use (Jap J. Soc. Terminal Syslemic Diseases, 6: 151-154, 2000) for more than 40 years. Because glucan is most commonly found in the walls of microorganisms, it represents a signal for the immune system of the human body to induce a non-specific immune response (Twig, V, et al. Orally-acting β1,3-glucan propliylactically protects against anthrax infection and cancer in J. Am. Nutraceutical Assoc 5 (2), 16-20, 2002). Glucan has traditionally been isolated from natural sources such as grain (especially oats, barley, rye: World Patent 200159147) and from cell walls of microorganisms. An important source of glucan is baker's yeast (Cz Patent 274918 and 281220) and higher fungal biomass (Borchers et al. Mushrooms, tumors, and immunity. Why Soc Exp Biol Med. 221 (4): 281-93, 1999). A major technological problem in the isolation and purification of pure glucan is the amount of other substances accompanying glucan in the starting natural materials (Autio et al. Effects of processing on the microstructure of oat bran concentrate and the physicochemical properties of isolated beta-glucans Food structure 11, 47-54 , 1992). This problem can be solved with the help of microorganisms that produce soluble glucan into the fermentation broth during submersive cultivation. In 1991, Lawford and Rousseau Bioreactor published a study on the fermentation preparation of very pure exopolysaccharide (glucan) in sub-culture using Alcaligenes faecalis and Agrobaclenum radiobacter. Overproduction (Buck et al. J. Gen. Microbiol. 51, 337-352 1968; Pazoutova et al., J. Nat Prod. 44, 225-235, 1981) of glucane in submersive cultivation of Claviceps sp. . accompanied by alkaloid biosynthesis. The indisputable advantage of using fermentation preparation is the relatively easy isolation of glucan by precipitation of acetone or ethanol from the biomass-free culture fluid compared to the complicated technology of isolating glucan from grains (Bhatty R. Extraction and Enrichment of (1-3), (1-4) -Beta- D-glucan from barley and oat brans Cereal Chem., 70, 73-77 1993). However, by precipitation with the solvent, the glucan is permanently converted into an insoluble amorphous form.

SUMMARY OF THE INVENTION

The present invention provides a method for the fermentative preparation of extracellular glucan using fungi of the genus Claviceps, wherein the producer uses a strain of the genus Claviceps which produces exopolysaccharides and does not produce alklaoids and related biologically active substances, especially from the group of strains Calviceps viridis and Claviceps sulcata. Submerged culture is performed at 18-28 ° C, on a synthetic culture medium, the biomass of the producing microorganism is removed from the culture fluid after fermentation by filtration or centrifugation, and the retentate is stabilized to the final form by lyophilization after dialysis.

In cases where the solubility of the glucan is not a prerequisite for application, this product can be isolated using a modified electrocoagulation method into a non-amorphous form. (Mollah et al., Electrocoagulation (EC) - science and applications J Hazard. Mat. B84 29-41 2001)

The method is to expose the retentate to direct current in an electrocoagulation device with an iron or aluminum sacrificial electrode at a current density of 0.05-0.8 mA / cnr of embedded electrode area at a voltage gradient of 0.2-2V / cm distance between the two electrodes.

This procedure can be modified by using stainless steel electrodes in the electrocoagulation apparatus and adding heavy metal salts, preferably aluminum, copper, iron, cadmium and lead salts in a concentration of 2-15 ml of saturated salt solution per liter of culture. fluid. In this way, a homogeneous mixture of spherical nanoparticles of different diameters can be prepared in conjunction with the effect of direct current.

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Soluble glucan was prepared by submersal ferinite with the help of strain Clavlceps sulcata PF2 from the collection of the Institute of Microbiology AS CR. The strain used does not produce alkaloids. Fermentation was performed in a 1 liter BioFlo culture apparatus (New Brunswick Scientific, Edison USA) at 24 ° C, 5 Hz stirrer speed and 0.3 WM aeration. T2 broth was used for cultivation (Pazoutova et al., J. Nat. Prod. 44, 225-235, 1981) and the fermentation was carried out in a batch process without automatic pH adjustment for 8 days. After centrifugation of the biomass, the culture fluid was freed of salts and residual sugar using a dialysis tube (Sigma Diagnostic, StLouis, USA) and dried in a service lyophilization unit BT MBU AS CR. The yield of soluble glucan was 8.3 g / L culture fluid.

Example 2

The culture fluid obtained by the procedure described in Example 1 was poured into an electrocoagulation device with two iron electrodes and the solution was subjected to direct current for 45 minutes (potential gradient 0.8 V / cm, current density 0.2 mA / cm ² area) After this time, 95% of the glucan was transferred to an insoluble form and, in the final step, separated by centrifugation on a laboratory centrifuge and after washing it was dried to a constant weight at 60 ° C.

Example 3

Submerged cultivation of Claviceps viridis strain in an MBR fermenter (Sultech, Switzerland) with a working volume of 7 liters at 24 ° C, stirrer speed of 2 Hz and aeration of 0.3 WM was performed. CS2 broth (Pazoutova et al., J. Nat.Prod. 44, 225-235, 1981) with an initial sucrose content of 50 g / l was used for cultivation. The fermentation was carried out in a batch process without automatic pH adjustment for 7 days. The strain used does not produce alkaloids. The culture fluid was freed of biomass by filtration on a vacuum filter. A laboratory apparatus for electrocoagulation was used as in Example 2 equipped with stainless steel electrodes (potential gradient of 0.8 V / cm, current density 0.24 mA / cm ² of embedded electrode area) and 5 ml / liter of saturated copper sulfate solution was added to the solution. After 60 minutes of direct current, all of the glucan was converted to insoluble glucan containing chemically bonded copper as a homogeneous suspension of microspheres with a diameter of 3-5 µιη. The total yield was 10.3 g / l of fermentation broth.

Industrial applicability

The soluble glucan preparation prepared according to the invention can be used as a food preparation with an immunoprotective effect, in the manufacture of paper and in the preparation of biodegradable polymer composites. The insoluble glucan isolated by electrocoagulation can be used to prepare nanocomposite membranes (E. Coronado et al. Nature 408, 447 2000) suitable for use in ultrapermeable membrane separation units.

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Claims (4)

Process for the preparation of extracellular glucan by means of fungi of the genus Claviccps, characterized in that the producer is using a strain of fungi of the genus Claviceps which produces exopolysaccharides and does not produce alklaoids and their like biologically active substances, in particular from the group of strains Claviceps viridis and Claviceps sulcata, and submersible cultivation is carried out at a temperature of 18-28 ° C, on a synthetic culture medium, the biomass of the producing microorganism is removed from the culture fluid after fermentation by filtration or centrifugation, and the retentate is stabilized to the final form by lyophilization after dialysis. Method according to claim 1, characterized in that the retentate is exposed to direct current in an electrocoagulation device with an iron or aluminum sacrificial electrode at a current density of 0.05-0.8 mA / cm ² of embedded electrode area at a voltage gradient of 0.2 -2V / cm distance between the two electrodes. Method according to claim 2, characterized in that stainless steel electrodes are used in the electrocoagulation device and heavy metal salts are added to the solution. Method according to claim 4, characterized in that 2-15 ml of a saturated solution of salts, in particular of aluminum, copper, iron, cadmium, lead, are added to the electrocoagulation device per liter of culture fluid.