CS245339B1 - Method of yeast biomass preparation from 1,6-anhydro-beta-d-glucopyranose - Google Patents

Description

The present invention relates to a process for preparing yeast biomass from 1,6-anhydro- [beta] -D-glucopyranose. The principle of the preparation of yeast biomass from 1,6-anhydro-4-D-glucopyranose consists in that the sterile liquid culture medium contains 2.0 to 6.0 wt. 1,6-anhydro- [beta] -D-glucopyranose is inoculated for 46 to 50 h with a culture of Aureobasidium pullulans CCY 27-1-14 or CCY 27-1-22 or CCY 27-1-32 or CCY 76-1-1, or CCY 103 and cultivation is performed at 25 to 28 degrees Celsius with agitation for 2 to 7 days. The invention has application in agriculture, food, chemical and pharmaceutical! industry.

The invention relates to a process for the preparation of yeast bomomase from 1'-anhydro-4-D-glucopyranose.

1,6-Anhydro- [beta] -D-gluicopyranose (levoglucosan) has not been used to produce microbial biomass to date because commonly used yeasts such as Saccharomyces cerevisiae, Saccharomyces vini and Candida utilis are unlikely to synthesize the constitutive or inducible metabolism of the required enzyme A. Kocková-Kratochvílová: Yeasts and yeast-like microorganisms, Bratislava, Alfa, 348 (1982)] Therefore molasses containing sucrose as the main sugar component is used to produce yeast biomass [D. Halama: Technical Microbiology, Bratislava, SNTL, 201 ( However, levoglucosan added to the culture medium in the form of a crystalline substance is used for the growth of Aureobasidium pullulans and other yeasts and yeast microorganisms such as Wingea robertsii, Arthroascus javanensis, Cephaloascus albidus, Candida ingens, Wickerhamia fluorescensomensensensensensususcescensomensens, , Pac hysolen tannophillus, Leucosporidium scottii, Trichosporon beigelii, as well as other β-glucosidase-producing microorganisms.

The Aureobasidium pullulans CCY 27-1-14 strain was isolated from the flowers of the Crathaegus oxyacantha plant from Devin Kobyla. Aureobasidium pullulans strains in the form of vultures with blastoconides having a length of 6 to 16 μτη (diameter 12.16 μΐη) and a width of 3 to 8 µm (diameter 4.45 µm). It forms hyphae of two species: air thin with a diameter of 4 to 6 μΐη and submerged thick with a diameter of 6.6 µm. The length ratio of blastoconidia is 2.73 and the surface quotient to cell volume is 1.12. Blastoconidia are spindle-shaped, which bud bilaterally and monompolarly forming both secondary blastodonidies that are smaller than primary blastoconidia [A. Kočková-Kratochílová, M. Černáková, E. Sláviková: Foil Microbiol. 25, 56 (1980) j. CCY strain 27-1-14 belongs to a group of strains which utilize D-xylose, L-arabinose, maltose, sucrose, lactose, cellobiose, trehalose, raffinose, melezitose, soluble starch, D- glucuronic and D-galacturonic, nitric acid and L-lysine. The CCY 27-14 strain utilizes atmospheric nitrogen. It grows good on sulphite liquor. CCY 27-1-14 strain produces enzymes such as urease (EC 3.5.3.1 J, extracellular rihonuclease, deoxyribonuclease, arylesterase (EC 3.1.1.2), endo-1,4-, β-D-glucanase (EC 3.2.1.4) ) and endo-1,4 -? - D-xylanase (EC 3.2.1.8) The enzyme monophenol monooxigenase (EC 1.14.18.1) only produces in trace amounts, so that, under given culture conditions, the fermentation medium does not blacken melanin production, such as CCY 27-1-14 produces polysaccharide pulullan and grows predominantly in the form of yeast cells [M. Černáková, A. Kocková-Kratochvílová, L. Suty, J. Zemek, L. Kuniak: Folia Microbiol., 25, 68 (1980), A. Kock-Kratochvil, L. Hronska: Folia Microbiol. 26, 117 (1981); A. Kock-Kratochvil: Folia Microbiol. 27, 404 (1981)].

Strain CCY 27-1-22 grows predominantly in the form of mycelia. They form polysaccharide pululan. Strain CCY 27-1-32 forms a black pigment melanin deposited mainly in chlamydospores. At the same time it produces pulullan. The CCY 76-1-1 strain consists of high-fat hyphae and further synthesizes heteropolysaccharide with considerable D-xylose content. It does not synthesize melanin. Strain CCY 103 grows in the form of chain cells and accumulates glycogen and trehalose. It does not form melanin and does not grow in hyphal form.

The principle of the preparation of yeast biomass from 1,6-anhydro- [beta] -D-glucopyranose in sterile liquid culture medium is that the sterile liquid culture medium contains 2.0-6.0% by weight of the composition. 1,6-anhydro- / β-D-glucopyranose is inoculated for 46-50 h with a culture of Aureobasidium pullulans CCY 27-1-14 or CCY 27-1-22 or CCY 27-1-32 or CCY 76-1-1 or CCY 103 and culturing is carried out at 25 to 28 ° C with stirring for 2 to 7 days.

An advantage of the proposed method of preparing yeast biomass from levoglucosan as the sole source of carbon and energy using strains of the yeast microorganism Aureobasidium pullulans is that it utilizes a substrate arising from cellulose waste. A further advantage is that Aureobasidium pullulans have such morphogenetic and biochemical properties that, together with its simple cultivation and production of sufficient biomass, are decisive factors for the industrial application of the biomass preparation process.

Example 1 h by culture of the strain Aureobasidium pullulans CCY 27-1-14 (the strain is deposited in the Czechoslovak Collection of Yeasts and Yeast Microorganisms, Institute of Chemistry, Chemical Research Center, SAS, Dubravska cesta 9, 842 38 Bratislava) is inoculated with sterile liquid culture medium containing 2 0.02% by weight of crystalline levoglucosan, 1.0 to 2.0% by weight of ammonium sulfate, 0.02 to 0.05% by weight of magnesium sulfate, 0.1 to 0.25% by weight of potassium dihydrophosphate, 0.025 ml of suspension microelements (the microelement suspension contains 0.1% by weight of ferrous sulphate heptahydrate, 0.016% by weight of manganese sulphate tetrahydrate, 0.0072% by weight of ammonium molybdate tetrahydrate, 0.0352% by weight of disodium tetraborate decahydrate, 0.006% by weight of copper sulphate pentahydrate , 0.124% by weight of ammonium sulfate heptahydrate) and 0.1 ml of a vitamin solution (the vitamin solution contains

0.1 wt. (1,2,3,5) 4,6 (cyclohexanedexole) (myo-inositol), 0.04% w / w; % pyridine-3-carboxylic acid (nicotinic acid), 0.04 wt. 4-aminobenzoic acid, 0.0002 wt. % Of 5- (2-oxoimidazolidino [4,5-c] thiol-2-yl) pentanoic acid, 0.04 wt. % D- (+) -N, N (2,4-dihydroxy-3,3-dimethylbutyryl) -β, β-2-aminopropionate (calcium pantothenate), 0.04 wt. % 3-hydroxy-4,5-bis (hydroxymethyl) -2-methylpyridine (pyridoxine), 0.02 wt. 3- (4-amino-2-methyl-pyrimidyl-5-methyl) -5- (2-hydroxyethyl) -4-methylthiazole (thiamine). The pH of the culture medium is adjusted to 5.0-6.5 with sodium hydroxide. Cultivation of the microorganism is carried out for 2 to 7 days at 25 to 28 ° C on a rotary shaker (2.5 Hz), the yield of yeast biomass reaching 0.9% by weight of dry matter, which is 45% / 1.6% by weight. Yeast biomass containing 37 wt% protein, 3 wt% ribonucleic acid, 0.4 wt% deoxyribonucleic acid and 59.6 wt% of the cell-related cell dry matter is separated from the culture medium media by centrifugation or filtration.

Example 2

The procedure was as in Example 1 except that 3.0 wt. % crystalline levoglucosan and as a nitrogen source 0.1 wt. potassium nitrate and such culture medium are inoculated for 48 h with a culture of Aureobasidium pullulans CCY 27-1-22 deposited in the same collection as CCY 27-1-14. The use of potassium nitrate as a nitrogen source increases the production of blastoconidia and suppresses the formation of bugs, so that the curl has a yeast-like character. Yeast biomass yield is 1.5% by weight. dry matter, which corresponds to 50% wt. 1,6-anhydro-trans-D-glucopyranose.

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Example 3

The procedure was as in Example 2 except that the culture medium containing 4.0 wt. the crystalline levoglucosan is inoculated for 46 h with a culture of the Aureobasidium pullulans CCY 27-1-32 strain deposited in the same collection as the CCY 27-1-14 strain. Yeast biomass yield is 2.1% by weight. dry matter. '

Example 1 and d 4

The procedure was as in Example 1 except that 5.0 wt. levoglucosan and as a source of vitamins 1.0 wt. the corn steep liquor and such culture medium are inoculated with a culture of Aureobasidium pullulans CCI 76-1-1 strain deposited in the same collection as CCY 27-1-14 for 50 h. Yeast biomass yield was 3.0 wt. dry matter.

Example 5

The procedure was as in Example 1 except that 6.0 wt. crystalline levoglucosan and as a source of vitamins 0.3 weight percent, yeast extract and such culture medium are inoculated with a culture of the Aureobasidium pullulans CCY 103 strain deposited in the same collection as the CCY 27-1-14 strain for 50 h. Yeast biomass yield is 2.9% by weight. dry matter.

Example 5

The invention can be used for feed purposes as a source of raw protein for animal nutrition or in obtaining various biofactors from yeast biomass, such as enzymes and polysaccharides useful in the food, chemical and pharmaceutical industries.

Claims (1)

A process for the preparation of yeast biomass from 1,6-anhydro-β-D-glucopyranose in a sterile liquid culture medium, characterized in that the sterile liquid culture medium contains 2.0 to 6.0% by weight of the composition. 1,6-anhydro- [beta] -D-glucopyranose is inoculated for 46-50 h with a culture of Aureobasidium pullulans strains CCY 27-1-14 or CCY 27-1-22 or CCY 27-1-32 or CCY 76-1-1- or CCY 103 and culturing is performed at 25-28 ° C with stirring for 2 to 7 days.