CS196162B1 - Solid cultivating medium inducing the intensive production of conidies at fungi of aspergillus niger type - Google Patents

Description

SOCIALISTIC AND DESCRIPTION OF THE INVENTION OF THE CERTIFICATE 196162 (H) (Bl)

/ 22 / Registered 24 11 77/21 / / PV 7759-77 / (51) jint. Cl.3C 12 N 1/16

ÚftAD pro inventzyA OBJEVY (40) Published on 06 06 7 9 (45) Published 15 06 8 2 (75)

Author of the invention LEOPOLD JINDŘICH doc. Dr. dr. ing., PLZEŇ and ULC JAROSLAV ing., ČERNICE (54) Solid culture medium inducing intensive conidia production in the fungus Aspergillus niger 1

BACKGROUND OF THE INVENTION The present invention relates to intensive production of fungi in such fungi of the Aspergillus niger species, which are difficult to form on the known solid sporulation soils, or spores cannot be obtained from these soils, using mineral sporulating soils containing in addition to the carbon source. and energy glycerol or glycerol and citric acid.

Tnyceliuro fungi belonging to the species Aspergillus niger can be cultured in the laboratory after inoculation of the fructifying organs / conidia / or vegetative parts of the fungus / mycelial fragments in a relatively small amount into the culture medium. However, if a fungus is used to produce a metabolite, such as citric acid, gluconic acid, and the like, on a commercial scale, only the conidia of the production fungus may be used as the inoculant, and must be available at each start of the fermentation cycle in sufficient quantities. . Under certain conditions, conidiophoryes of sterigma, at the ends of which conidia chains are formed, are believed to grow as so-called heel cells.

In order to obtain conidia by scraping or aspiration, the cultures in question are cultured on a solid, i. The assumption of relatively fast, but intensive conidia production is a certain culture / sporulation / soil composition, which is sometimes specific for individual Aspergillus niger strains. For the Aspergillus niger and other industrial strains, a number of regulations have been developed for the composition of sporulation soils, which have been published in Part I in the literature (for example, by C. Booth, Methods in microbiology, Vol. 4, Acad.Press, London). -New York 1971; W. F. Harrigan, M.F.

Mc Cance, Laboratory Methods in Microbiology, Acad.Press London-New, York 1966; G.SmithAn odutroduction to industrial mycology.6.Ed.Arnold Ltd. London 1969. Typically, nutrient-poor media are used that favor sporulation rather than rich media. Several special sporulation media for Aspergillia are recommended by Moyer, Wells, Stubbs, Herrick, and Mey / cit.dle Thom C.M. and R.B.Raper, A. The Williams- Wi lkins Comp., Baltiraore 1945 /. This is usually a material medium containing potassium (sometimes sodium), magnesium, phosphate, nitrogen (usually in the form of ammonium nitrate) and glucose with the addition of malt or glycosylate; other sporulating media contain sucrose and nitrogen instead of glucose in the form of peptone. Most strains of Aspergillus niger on these media produce very dense and uniformly sporulating cultures whose considerable amount of conidia is readily obtainable by scratching or aspiration.

However, Aspergillus niger fungi are found to be dissatisfied with these proven sporulation agar soils; sporulation is sparse, uneven, the heads are small and conidia are difficult to obtain or are difficult to remove as the culture touches the scraper or tube of the vacuum cleaner. In other cases, the fine-celled layer carrying conidophors, crabs, so that conidia cannot be obtained by scratching, as mycel tears from the agar. Tako- 196162

Claims (3)

The disadvantage is very serious, since the inoculum material is insufficient to apply the culture in question, even though it has the best production ability. The abovementioned drawbacks of conventional solid sporulation instincts, manifested in the cultivation of certain strains of Aspergillus niger, have been eliminated in the agar sporulatory compositions of the present invention, in addition to malt / glycerol, sometimes citric acid as carbon and energy sources. Alcohols were tested with one / methanol, ethanol / and two / ethylene glycol / OH groups, which did not have a fungal effect on the fungus; only glycerol, sorbitol and mannitol were effective. Among the organic acids, sporulation of two carboxylic acid groups (malonic acid, succinic acid, glutaric acid) and with three carboxyl and hydroxy groups (lactic acid, malic acid) and three tri-carboxylic acid groups and hydroxy-group (citric acid) acted favorably. On soils that contain one or two 2 tec / hecto compounds, non-scrambled, mycelium-bound, myceles with innumerable conicides with black heads grow up and can be scraped off without difficulty. The following examples are given as examples: 'The chemicals are pure for analysis, the malt extract is stored in the refrigerator, otherwise it soon starts to ferment and is useless /: Glycerol / 100% / 105 g / rfsp. this quantity corresponds to the amount of less concentrated glycerol / malt extract / 80% / 45 g ΝχΝ03 0.45 g MgSO4-7H2O 0 ^ 06 g NaCl 15 CuSO ^ .Sl120 0.001 5g agar 30 g Make up to 11 with distilled water niger may omit the addition of copper sulphate. The strains of A. niger fungi are preferably supplemented with 4% citric acid, partially neutralized with KOH, so that the pH of the soil is 4.8 to 5.0, ensuring that the soil solidifies upon cooling. The conidia obtained from the agar soils of the present invention can be used for deep fermentation as such or for surface fermentation with sterile activated carbon or sterile calcium carbonate or both. Both conidia germinate in both cases and the yields of citric acid remain unaffected by the cultivation of conidia on the agar media of the invention. From a conserved Aspergillusniger production strain, we transfer a small amount of conidia to a suitable liquid sporulation soil and only A solid culture medium inducing intense conidia production in fungi of the species Aspergillus niger, comprising as a carbon source a malt extract, characterized in that it contains 8 to 12% by weight of a polyhydric alcohol in the range of 3 to 6 C atoms and / or 1 as an additional source of envelope. up to 5% by weight of polyhydric acid or hydroxyethyl in the range of 3 to 7 C atoms. 4, the conidia produced here serves to inoculate the sporulation agar of the present invention. This process greatly extends the viability of the canned Aspergillusniger strain. Agar sporulation soil according to the invention was tested in a number of different Aspergillus niger strains and proved to be 60% of the harvest. The following are examples of sporulation-agar media according to the invention. The morphological appearance of the cultures, the ease of conidia reduction from the cultures and their biochemical activity by fermentation experiments on citric acid production by inoculation on the yeast-phase substrate and the determination of citric acid after the end of the fermentation experiment were examined. EXAMPLE 1 Dissolve a quantity of glycerol corresponding to 105 g of 100% glycerol in distilled water, followed by 45 g of malt extract (with 80% dry matter), 0.45 g of ammonium nitrate, 0.06 magnesium sulphate / with 7 H2O / 15 g of chlorine. of sodium sulfate, 0.0015 g of copper (II) sulfate (with 5 H2O), 30 g of agar and added to 1 L with water. The solution is boiled in an enamelled flask until the agar is boiled, the evaporated water is added and the agar solution is poured, for example, into 100 ml of 300 ml Erlenraeyef flasks which are sterilized at 0 DEG C. for 1/2 hour after closure with pads. 5, or in trays, the agar soil height should not be less than 2 mm. After cooling, the solidified agar medium was inoculated in a Hansen box with Aspergillus niger conidia and incubated for 8-10 days at 32 ° C. On the surface of the thin white mycelium, a dense stand of high conidiophores bearing dense carpet-like heads with innumerable conidia, which can be easily scraped or aspirated, is formed. After inoculation on the fermented molasses solutions, normal, folded and white mycelia have grown and, after a weekly the fermentation yield of citric acid was 77.5% on average; sugar. EXAMPLE 2 An agar sporulation soil was prepared as in Example 1, but was filled with g citric acid and an amount of potassium hydroxide to make the pH of the soil about 5. Otherwise, the procedure was as in Example 1. When sterilizing, care should be taken to ensure that agar flasks were removed from the autoclave as soon as possible after the completion of the sterilization; otherwise, there is a risk that the agar medium will soften. The appearance of A. niger fungi was, after inoculation and incubation at 32 °, the same as Example 1. The conidia could be easily scratched. In fermentation experiments, the average citric acid yield was 78.1%, calculated on seeded sugar. THE INVENTION 2. Solid culture medium according to claim 1, characterized in that glycerol, sorbitol, mannitol is used as polyhydric alcoholyl. 3. Solid culture medium according to claim 1, characterized in that malonic, succinic, glucaric, lactic or citric acids are used as organic multivalent acids or hydroxy acids. & * vrro £ rafiA- p