CS201693B1 - Strain of micro-organism saccharomyces cerevisiae - Google Patents

Description

The invention relates to a strain of Saccharomyos cerevisae CCY 21-4-2l-e which produces above 2% ergosterol in dry matter biomass.

Saccharomyces yeasts are the most important ergosterol producers. The values reported in the older literature are relatively low. Proakurjakov, Popova and Ossipov (Biochimia 3: 397, 1938) found 0.31% for Monilia murmanica, 0.55% for Torula utilis, and 2.36% for Frohberg breeders. Therefore, they consider brewer's yeast to be particularly rich in ergosterol, e.g. even strain 776 had 2.5 wine yeast had only 0.4 to 2.19% ergosterol in dry matter. Appleton and Kleber (Appl. Microbiol. 3: 249, 1955) demonstrated only 0.36% in Candida krusei and 0.06% in C. parapsilosis, but up to 9% in S. cerevisiae, total sterols. Mitra and Garg (J. Inst. Brew. 64: 327, 1958) determined 0.1-0.5% ergosterol in dry matter in the Dhar yeast, which they considered equivalent to other assays in Terula utilia. For more than 20 years, he has remained in a relative state of tranquility in vitamin D, and has only increased in the last few years. Jeliazjan, Marojan and Arumjunian (Microbiology 44: 632, 1975J) determined the total atherols content of yeast and confirmed that the highest production is in carbohydrates, from 1.3 to 3.13% per biomass dry matter. Microbiol. (11:21, 1975) found ergosterol levels in Candida, Torulopsis and Rhodotorula and found to be between 0.4 and 0.6% on dry matter, and found only 2.4% in Saccharomyces carlsbergensis 4228. Recent Kosikov, Lyapunova (Microbiology 46:86, 1977) report the production of ergosterol in yeast as follows:

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201 093 bakery yeast Odesskaja 14 produces 2.3%, indeed baker's yeast from 1.8 to 1.9%, distillery breeds from 2.0 to 2.2% and brewery race 4228 around 1.5% ergosterol in dry matter.

From these cultures, the monosporic cultures were isolated, lira, haploid and diploid, and a number of hybrids that produced from 1.3 to 2.3%. They managed to obtain adenine deficient mutants in which ergosterol production was increased up to 4.8% in dry matter.

The present invention provides a strain of Saccharomyces cerevisiae CCY 21-4-21-e that produces a large amount of ergosterol. The wild-type 21-4-21 strain, which accounted for only 10-25% of the cells and a P / 0 quotient of less than or equal to 1, was inoculated in suspension! 10 cells / ml per wort agar surface and a camphor crystal was deposited in the center of the agar surface dish. Under the influence of this mutagen, thorn culture was carried out at 28 ° C until a unique yeast colony appeared on the surface. By repeated sowing on camphor plates, a mutant strain was produced that produced only cells with a surface / volume coefficient of less than 1 (towards the ends of the exponential phase, i.e. at the time of maximum ergosterol production). The strain thus bred was designated as CCY 21-4-21-e.

The CCY 21-4-21-e strain produces round, oval and elongated cells whose P / 0 quotient is less than 1. The enormous colony of this strain is smooth and increments by about 1.7 mm in 100 hours. In a liquid environment, the strain sedimentes quickly, forming no skin, wax or ring on the surface.

The CCY 21-4-21-e strain does not spore after induction with acetate. It needs vitamins to grow. At 5 ° C it grows very weakly, at 42 ° C it grows well, but optimally the growth temperature is 28 ° C. Ferments glucose, galactose, meltose, sucrose and raffinose. It also assimilates these sugars and, in addition, utilizes maltotriose and maltotetraose, but does not assimilate potassium nitrate or lysine. It breaks down sucrose within 3 hours and glucose and fructose and then completely utilizes these monosecharides within 12 hours. In grape raisin, in beer wort and in molasses environment, raetie with the same speed. It can withstand up to 12% ethanol and 5% sulfuric acid. It is sensitive to actidion at a concentration of 0.1 µg. 2% lactic acid inhibits culture growth by 60%, 2% tartaric acid by 15%. Extracellular polysaccharides give an iodine-starch reaction.

CCY 21-4-21-e strain does not agglutinate with serum prepared against typical representatives of Saccharomyces cereviciae or only at the lowest dilution titer. It agglutinates with serum against Candida robusta and Saccharomyces cartilaginpsus at a dilution titre of 320. Candida robusta and Saccharomyces cartilaginosus are now counted as Saccharomyces cerevisiae and appear to be marginal variants of this species in numerical classification.

The CCY 21-4-21-e strain was grown on Olson-Jensen artificial composition and induction molasses soil on a shaker and continuously. The composition of p6d and the results of the culture can be seen from t.2.

Ergosterol was determined after culture and ly, of ili alebo cation or directly using wet biomass. A 50 to 100 mg sample of lyophilized yeast was used for the assay, which was hydrolyzed in 40% potassium hydroxide for 1 hour at 0.196 MPa, shaken with diethyl ether for 2 hours on a shaker ppi 4 ° C. After removal of the ether layer, the aqueous layer was shaken again with ether and the combined ether extracts were washed well with distilled water.

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The third ether extract ea was evaporated to dryness in a rotary evaporator. The residue was dissolved in 0.5 ml of chloroform and made up to 10 ml with ethanol. diluted with chloroform: ether 5: 100. The detection of ergoeterol aa was checked with a registration spectrophotometer and the amount of e was calculated from hours at 282 nm.

Tab. 1 shows the dependence of ergosterol formation on the genotype of colony cells, which was verified by the morphological characterization of the cells of the population as mentioned above. The dependence of ergosterol formation on the average quotient of P / 0 is directly proportional in a region of about 1 and above, in a P / 0 region of less than 1 it is no longer proportional because P / O is not usually less than 0.7. Here the external influences stimulate.

Thus, the OCX 21-4-21-e strain has found many different values of ergosterol content in yeasts ranging from 2.69 to 4.35 (with an average P / 0 of less than 1) an average of 3.64% ergosterol in dry matter, standard deviation 0.52 and coefficient of variation 14.32.

Table 2 shows the results of the experiment and cultivation of the OCX 21-4-211-e strain under various conditions, i. on the soil of different composition in both static and continuous cultivation. These results indicate that the ergosterol content of the selected strain 21-4-21-e may even exceed 6% in dry matter. The biomass increments on the melase soil are satisfactory.

Table 3 shows a comparison of the mutant properties with the wild-type strain.

Table 1

Dependence of ergosterol production on the morphological character of the cell population in OCX 21-4-21

population number Average quotient P / 0 % ergosterol in dry matter biomass (average 2 to 3) replicates) e / 3 1.00 2.69 e / 6 1.15 2.24 e / 2 1.30 2.23 e / 4 1.42 2.15 E / 1 1.44 2.12 e / 7 1.47 2.10 e / 8 1.70 1.85

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Table 2

Biomass and ergosterol production under different cultivation conditions

Attempt δ. Growth on Olson-Jensen ^x mg dry matter.ml ” ¹ after 24 h Induction molasses p6da ^XX mg dry matter.ml ” ¹ after 7 2 h Ergosterol production in% of biomass dry matter na trepaSke 48 h after 72 h continuously after 48 h after 72 h 1 2.9 8.7 5.9 6.2 5.1 6.2 2 2.6 8.8 6.0 5.3

^x Olson-Jensen composition: 1.1 g KHgFO *, 0.5 g MgSO ₄ , 1.5 g esparagine, 0.1 gOaCl 2,

1.5 g of sodium citrate, 1.0 powder of yeast extract and 20 g of glucose per 1,000 ml of distilled water. Add 1 ml of solutions A, B, S.

Solution A: 0.08 g of acid. folic acid, 0.5 g m-inoeitol, 0.44 g thiamine, 0.15 g acid. nicotinic

0.05 g Ca-pantothenate, 0.120 g pyridoxine per 100 ml distilled water

- Solution B: 0.25 g ribovlavin, 0.3 g kye. p-aminobenzoic acid, 0.05 g biotin per 100 ml dest.

waters

Solution S: 0.146 ZnSO ₄ , 0.067 g CuSO ₄ , 1.18 g Fe (NH ₄ ) ₂ SO ₄ per 100 ml dist. water.

^ Composition of induction molasses: 102 g molasses, 10 g corn steep, 0.06 g (NH4 JgHPO4)

Dissolve 0.5 g of KgHPO4 in 0.5 liters. 1% aluminum sulfate. Stir aa for 20-25 min, neutralize NaOH to pH 7, boil, filter and re-adjust pH to 7 *

Table 3

Comparison of some mutant properties and wild-type strain

property Wild tribe CCX 21-4-21 mutant OCX 21-4-21- · Morphological parameters on wort after 3 days, 28 ° 0: Avg: cell length 7.39 14.84 length-width ratio 1.16 1.18 cell surface / volume ^1.15 0.70 Flocculation ability by count cells in sounds (average) 7 1 to 2 The character of a huge colony oily shiny, African shine rough, edge straight edge Oamofilia lobed 40% 28% (Bear) sucrose sucrose

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Claims (1)

Saccharomyces cereviaiae strain CCY 21-4-21-e producing ergosterol.