SU1521767A1 - Method of obtaining hybrid strain saccharomyces cervilae for fermenting starch - Google Patents

Abstract

The invention relates to the baking, alcohol and brewing industry, in particular to technological processes associated with the disposal of starch-containing raw materials. The aim of the invention is to obtain strains containing an expressing glucoamylase gene. A method for producing hybrid strains of yeast fermenting starch-containing raw materials is to create hybrids of the yeast SACCHAROMYCES CEREVISIAE, which contain genes that feed glucoamylase and are able to secrete this enzyme into the culture fluid. By means of the method, intensive fermentation of starch-containing raw material is achieved without saccharification prior to commercial amylolytic enzymes or malt. Hybrids are obtained by crossing the SACCHAROMYCES DIASTATICUS CBS 1782 strain carrying the glucoamylase structural gene and the SACCHAROMYCES CEREVISIAE strain carrying the glucoamylase repressor gene. The hybrids are then transferred to a nutrient medium containing sodium acetate to induce sporulation, after which they isolate spores capable of utilizing starch and cross them with the SACCHAROMYCES CEREVISIAE strain carrying the auxotrophy marker. Further, the operations are repeated in a predetermined order until hybrids are obtained with a sporulation efficiency of 90-95%. 1 tab.

Description

The invention relates to the baking, alcohol and brewing industries, in particular, to the technological process associated with the utilization of starch-containing raw materials using food yeast Saccharomyces cerevisiae.

The purpose of the invention is to obtain strains containing an expressing glucoamylase gene.

The method consists in obtaining strains of the yeast Saccharomyces cerevisiae, containing the structural glucoamylase gene and the inactive allele with

its repressor and capable of using starch as the sole carbon source, secretes glucoamylase into the medium, while simultaneously fermenting the glucose produced. Proposed yeast strains Saccharomyces cerevisiae obtained as a result of several stages mating yeast Saccharomyces diacta- ticus CBS 1782 glgoko- secreting amylase, yeast Saccharomyces cerevisiae, from Peterhof genetic lines leading th tsenie and origin of race S, cerevisiae XII, as well as with yeast - saharomitsntam from Karboischaylskikh- genetic lines. The proposed yeast strains S, ce. Revisiae have the following morphological and cultural characteristics.

Cells are oval, less often almost round, average size 6.7 x

X. 5.0 microns. Reproduction by budding.

After 96 hours, the incubators on the wort agar form round colonies with a diameter of 4.0 - 6.3 mm. The edge of the colony is flat or slightly striated, the surface of the mat. Yeast ferments glucose, fructose, sucrose, maltose, galactose, raffinose, starch; do not ferment xylose and arabinose. Gelatin is not diluted, glycerin and alcohol (ethanol) are absorbed. Do not assimilate mannitol, dulcite and sorbitol.

Cultures from the obtained genetic lines are in a liquid medium on a rocking chair or on agar media containing one of the following carbon sources: glucose (2%), maltose (2%), sucrose (2%) or ethanol (1.5%), and also peptone (1%) and yeast extract., (or yeast autolith) (0.5%) as a source of growth substances. Grown biomass

used for sowing in fermentation medium. The seed volume is (O, 5-5.0) -10 cells / ml. The broth medium has a pH of 5.0-6.0 and contains gelatinized starch (1.5-4.0%) or flour (4%). In case of use

starch growth media is introduced into the medium — yeast extract (0.5%) or yeast autolysate (0.5%). The fermentation is carried out at 28 - 35 ° C for 48-96 h. The fermentation process is controlled by an intensive spike.

The method is illustrated by specific examples.

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PRI me R. 1. The production of S. cerevisiae yeast lines is carried out in two stages.

Step 1. The genetic lines of S. cerevisiae are obtained as follows.

Cell culture of S. cerevisiae CBS 1782, secreting glucoamylase and having the genotype STA 2 sta 10 (STA 2 5

0

five

P

. Q

The Q allele, coding glucoamylase, sta 7 10 - inactive allele of the glucoamylase repressor gene), is grown on a full-fledged medium with glucose for 48 h. Then, using a micromanipulator, individual cells are laid out, which are laid out on a full-fledged agar, de, placed In these cells, vegetative cells of the strain S. cerevisiae No. 117 of the genotype sta 2 STA 10 (sta 2. - absence of the structural genes of glucoamylase, STA 10 - the repressor gene of glucoamylase). After 4 hours of incubation, the resulting zygotes are selected and, after the vegetative multiplication stage of these hybrids, sporulation is induced in them on an acetate medium. Hybrids have low sporulation efficiency and high dispute abnormality. Surviving segregants among spore descendants isolated by tetrad analysis are tested for the ability to synthesize glucoamylase, which manifests itself in the form of a starch decomposition zone around colonies growing on nutrient plates containing starch (1: 6%) and having a pH of 5 ,four.

Stage 2. Selected auxotrophic segregants have the STA 2 sta 10 genotype: ade 5. Then, they are followed by baking backcrosses for S. cerevisiae strain No. 134, which carries an auxotrophic muttach in the Sep 2 gene. descendants each time, cultures of the STA 2 sta 10 genotype were synthesized by glucoamylase. In the third generation, cultures were obtained containing the structural gene for glucoamylase and secreting glucoamylase in the medium. Such cultures intersect well with each other, as well as with S. cerevisiae type lines, giving hybrids with a spore formation efficiency of 93% and a spore survival rate of 84%. As a result, it was confirmed that genetic lines of the S. cerevisiae yeast of the indicated genotype, capable of secreting glucoamylase and utilizing starch as the only carbon source, were created.

five

0

five

5152

Example 2. Step 1. Obtaining lines is carried out as in Example 1.

Stage 2. Selected auxotrophic segregants have the genotype STA 2 sta 10 ade 5. Next, they carry out consecutive backcrosses to S. segusiae strain No. 1b40, which carries an auxotrophic mutation in the tt1 gene. At the same time, among descendants every time cultures of the genotype STA 2 sta 10 were synthesized by glucoamylase. In the third generation, cultures containing the glucoamylase structural gene and secreting glucoamylase into the medium were obtained. : Such cultures are intercrossed both with each other and with the S. cerevisiae lines, giving hybrids with a sporulation efficiency of 95% and a spore survival rate of 90%. As a result, it was confirmed that genetic lines of the yeast S. cerevisiae of the indicated genotype, capable of secreting glucoamylase and utilizing starch as the only carbon source, were created.

PRI me R 3. Step 1. Obtaining lines is carried out as in Example 1.

Stage 2. Selected auxotrophic segregants have the STA 2 sta 10 ade 5 genotype. Next, they are followed by successive backcrosses for S.uevisus No. 193 strain, which carries an aukrrotrof mutation in the yoke 3 gene. among the descendants, each time, cultures of the genotype STA 2 sta 10 are synthesized by glucoamylase of the third generation. Cultures containing the structural gene for glucoamylase and secreting glucoamylase in cpe were obtained in the third generation. Such cultures are well screened with both among themselves and with the S. cerevisiae type lines, giving hybrids with a sporulation efficiency of 90% and a spore survival rate of 66%. As a result, it was confirmed that genetic lines of the yeast S. cerevisiae of the indicated genotype, capable of secreting glucoamylase and utilizing starch as the only carbon source, were created.

Example4. Determination of glucoamylase activity.

The obtained cultures of S. cerevisiae determine the activity of glucoamylase. For this, cells are seeded in a complete liquid medium containing soluble starch (3%) and having a pH of 5.4. After 72 h of incubation with precipitation

Q 5 0 5

About Q 5 0

five

76

The cells are added to 1 part of the culture fluid with 1 h of a solution of amylase in dimethyl sulfoxide (50 mg / mp) as a substrate. In the reaction mixture thus prepared, glucose accumulation dynamics were measured using a glucose oxidase reagent. From this dynamic, the glucoamylase activity is calculated in Standard Units (μmol glucose / / ml min). For different cultures of the obtained genetic line, the activity is 0.03-0.05 units. The control group of S. cerevisiae XII does not possess activity.

PRI me R 5. The use of cultures from the obtained lines.

A set of 5-4A, 5-4B and 8-11C cultures related to the established genetic: KIM lines, as well as the control strain S. cerevisiae.XII, used for fermentation of starch, are grown for 48 hours in a dense nutrient medium containing glucose (2%), yeast extract (0.5%) and peptone (1%). After the specified period, the cells are seeded in flasks with a liquid medium containing starch (3%) and yeast extract (0.5%) and having a pH of 5.4. The seed volume is 1-10 cells / ml. The seeded flasks are incubated at 30 ° C without aeration to induce fermentation.

The table presents the results of measurement of cell titer and fermentation characteristics (based on CO release) over time.

From the table it is seen that hybrid strains of S. cerevisiae are able to carry out the fermentation process, as evidenced by the rapidly increasing release of CO2, accumulate biomass using starch, which is simultaneously saccharified by glucoam 1, synthesized by cells. At the same time, the strain S. cerevisiae XII used in the prototype method does not possess this ability.

Thus, the proposed method makes it possible to obtain new hybrid strains of the yeast S. cerevisiae capable of fermenting starch-containing raw materials without prior saccharification by amylolytic enzymes. This reduces the consumption of saccharifying enzyme commercial preparations or malt, and also eliminates the technological KHir stage of preliminary saccharification.

Claims (1)

Invention Formula A method of producing hybrid strains drozhlsey Saccharomyces cerevisiae / ViH starch fermentation, predusmat ™ rivayu1tsy crossing strain Saccharomyces diastaticus CBS 1782} glyukoamilazy8 bearing structural gene and the strain Saccharomyces cerevisiae, carrying the glucoamylase gene repressor, characterized in that, with In order to increase the strains containing the gene expressing the glucoamylase gene, obtained by crossing, the hybrid is transferred to a nutrient medium containing sodium acetate to induce sporulation, then isolate spores capable of utilizing starch, and cross them with the Saccharomyces cerevisiae strain, Hecyniji MI maker. Further, the operations are repeated in a predetermined order to obtain hybrids with a sporulation efficiency of 90-95%. (without starch saccharification)