Yeast strain saccharomyces cerevisiae used for obtaining alcohol

FIELD: biotechnology.

SUBSTANCE: alcohol (8-C) strain Saccharomyces cerevisiae No 8 having a high generative activity was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-3855 and can be used in production of alcohol.

EFFECT: invention enables to increase the alcohol yield and to reduce the formation of byproducts.

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The invention relates to the alcohol industry and represents a new strain of the yeast Saccharomyces cerevisiae In - 3855 used for alcohol production.

In the production of alcohol from molasses yeast should have a high fermentative energy with anaerobic type of breathing, carry a high concentration of salts and solids, to be resistant to the products of metabolism of foreign microorganisms.

In the production of alcohol from molasses are different strains of the yeast Saccharomyces cerevisiae and among them the most common is the production race I, with good technological properties [1, 2] is similar. However, this race has a relatively low generative activity, has increased the need for growth stimulants, sensitivity to contaminants molasses medium. In terms of morphology, the strain is non-uniform in size and shape, with the presence in the population of elongated cells having a slow growth rate, long generation time compared to oval cells.

It is also known that the traditional medium for growing yeast S. cerevisiae used in the alcohol industry is molasses culture medium. The creation of a modern progressive technologies food alcohol dictates the need for physiologically active RAS yeast, making the surrounding efficient digestion of concentrated molasses wort. We have found that the mineral and organic matter geothermal water nefrologia class can be considered as a new source of supply of yeast. It was also established that stimulation of the synthesis of ethanol at 25% and a significant reduction in undesirable impurity compounds using strain S. cerevisiae Y-503 due to the possibility of changes in the metabolism of yeasts on the basis of the influence of biologically active substances geothermal water [3, 4]. In this regard, a new strain get on molasses nutrient medium, where the mineral and organic food uses geothermal water nefrologia class with General mineralization of 5.2-5.4 g/l, diluted with tap water to a salinity of 4.0-5.2 g/L. the Presence of such important minerals necessary for vital activity of living organisms, as K, Na, Mg, Ca, Fe, Mn, as well as boric, silicic acid, organic matter, particularly humic, which are stimulants membrane rearrangements in living cells, create favorable conditions in the environment of the cultivation of for the cultivation of yeast organisms.

Object of the invention is the creation of stable alcohol yeast strain for efficient fermentation of molasses nutrient medium with the use of geothermal water nefrologia class with the concentration of carbohydrates 21.0 g / 100 cm3 that can save spirostachys, high biochemical and technological properties of a breeder with a long passirovannye it on solid and liquid media. The technical result is to increase the yield of alcohol.

The proposed strain of yeast has the following characteristics. Morphological and cultural characteristics. Cells in the daily culture of molasses nutrient medium with geothermal water (ESGV) are rounded-oval in shape, size 6-8×11-13 µm; NSGW + agar - oval, ovate, 6-7×11-12 μm. Culture propagated vegetatively, forming bags with spores (Wednesday Gorodkovoj, gypsum blocks, hungry agar). Micro-colonies on NSGW + agar - rounded, smooth, slightly shiny with smooth edge, size 4-7 mm, yellow color. Microcolony (20 daily)- rounded shape, size 2.9×2.7 cm, surface radially streaked with concentric circles, slightly shiny, pale, wavy edge, the profile is slightly convex with a small cone, homogeneous structure, consistency spotting (Fig.1,2).

Physiological and biochemical characteristics. The strain of S. cerevisiae In - 3855 fully sprayway: glucose, fructose, sucrose, lactose, maltose, raffinose, simple dextrins; not sprayway and do not absorb lactose and inulin. Absorbs acetic, lactic, is not absorbed succinic, malic, is nnow, citric acid [5]. Sprayway highly concentrated rasterops with carb 21.0 g / 100 cm3by synthesizing 12.4% vol. the alcohol. Optimum growth temperature is 20-30°C but can grow at temperatures of 32-33°C. the Optimal pH values of the environment are 4.5-5.5.

The proposed strain of S. cerevisiae In - 3855 stored in the PMBC Vniigenetika under the collection number In - 3855 and yeast collection of the Caspian Institute of biological resources of Dagestan scientific center of RAS, Makhachkala. Example of getting a strain.

The original culture to develop a strain of S. cerevisiae In - 3855 served strain of S. cerevisiae Y-503 [4]. To obtain yeast with the required properties was carried out adaptation of S.cerevisiae strain Y-503 to molasses nutrient medium to moderately mineralized geothermal, sulphate-chloride-sodium bicarbonate water from wells # 26 Makhachkala field. As the impact of nutrient medium composition microorganisms adequately changed, which led to the emergence of new forms. Significant importance and frequency of transfers to fresh medium.

In the first phase of research from numerous individual microcolonies (95), by finding useful morphological and physiological properties was screened active populations studied races. The result has been allocated 6 monocultures by sieving GRT the early clones on nutrient medium with high concentration of solids. Observation of the dynamics of the fermentation of all cultures revealed the identity of the release of carbon dioxide during the first 24 h after inoculation. Significant growth of fermentative activity was manifested in 3-8 days, which is more marked in 3 variants. In the second stage adapted yeast suspension were sown on thick molasses medium with geothermal water. Each grown colony was eliminated on the beveled agrisurance molasses wort. At all stages of the research monitored the functional morphology of yeast cells. Observations were conducted for speed and nature of budding, growth, reproduction, shape and size of cells. Most of them fermentation ability, the best morphological properties had culture - 3855. The ethanol concentration in the mash was increased by about 12.4 %. A comparative study of the dynamics of the fermentation of molasses wort concentration of carbohydrates 21.0 g / 100 cm3breeding strain In - 3855, the original Y-503 and industrial - I, showed the promise of using the selected strain.

The proposed strain is stored on the beveled NSGW+agar in the refrigerator at a temperature of 0±6°C.

An example of using the strain.

Cultivation of yeast using the proposed strain of S. cerevisiae In - 3855 carried out for 72 h deep m the mode in periodic mode under anaerobic conditions in a laboratory setting at a temperature of 30°C±1, pH 5.0 molasses nutrient medium with geothermal water (ESGV) and carbohydrate content 21.0 g / 100 cm3the following composition (g/l):

- molasses 488.74

- hydroalcoholic ammonium 2.58

- geothermal water with a salinity of 4.0-4.2 g/l with a defined qualitative and quantitative composition necessary for the cultivation of yeasts else

To add molasses diluted with tap water geothermal water content in the environment of carbohydrates 21.0 g/100 cm3hydroalcoholic ammonium - 2.58 g/l concentrated sulfuric acid to achieve in the medium pH 5.0, at the rate of 0.4-0.6 ml per 100 g of molasses. The contents mixed well. Sterile nutrient medium is poured into 1.5 liter vessels with a capacity of 3 liters, then seeded vegetative cultures of the indicated strains in the amount of 150 ml of yeast suspension last stage adaptation NSGV, 1 ml of which contains 95.6 million/ml cells. As antifoam use struktol 0.1 ml/1.5 l medium. After the 72-hour experiment, the yeast is separated from the culture fluid by centrifugation (5000g, 15 min on the stationary laboratory centrifuge SSC-344.2. Upon completion of the experiment in the fermented substrate is defined residual sugar, the accumulation of alcohol and the content of impurity compounds. The greatest number of alcohol noted in a sample of S. cerevisiae In - 3855 at minimal the nom content of residual sugar (table 1).

The absorption of raffinose given culture (proposed strain) occurs to a greater extent than the original S. cerevisiae Y-503 and production race I (1/3), which is caused by activation of the enzyme α-galactosidase, which catalyzes the digestion of carbohydrate. Apparently, the organic components of the geothermal water (humic acid), affecting colloidnochemical properties of protoplasm and the efficiency of the cell from the culture medium of biologically active substances, thereby increase the enzymatic activity of living cells.

It is established that a prototype has a higher level of activity of β-fructofuranosidase, which is evident from the first minutes of fermentation. Found that the level of activity of aldolase in strain S. cerevisiae In - 3855 exceeds the indicators of input and control at 6 and 25%, respectively. The key substance anaerobic breakdown of glucose and the metabolism of Sugars is pyruvic acid, the decomposition of which acetaldehyde and CO2catalyzed by piruvatcarboksilazy. The obtained data allow to speak about a higher activity of the enzyme experienced in living biomass on 27 and 37% compared with the original culture and control. The final stage of fermentation catalyzes alcoholdehydrogenase, healing acetaldehyde to ethanol. The activity of this enzyme in glue the hands of an experienced option S. cerevisiae In - 3855 much greater than the source and the control (table 2).

Analysis of the chromatograms of volatile impurities revealed identical qualitative composition and strain of S. cerevisiae In - 3855 differed by the number of metabolites synthesized - 30.18% less compared to S. cerevisiae Y-503, which is the result of changes in the regulatory functions of the cell (table 3). Thus, In S. cerevisiae - 3855 synthesizes acetaldehyde almost twice less than the strain S. cerevisiae Y-503. Acetone (29.16% less than in the experiment) can serve as a necessary product for the formation of higher alcohols, which may be undesirable for the production of the main product is ethanol. Using a culture of S. cerevisiae In - 3855 alcohols include propanol-1, Isobutanol, butanol-1, isoamyl and hexanol found in much smaller quantities compared to strain Y-503 (8518.24:10947.20 mg/DM3experience: source). To a greater extent the presence of propanol-1 and isoamylase in control at 30 and 14%, respectively, impair the technological characteristics of the substrate with acquired fusel smell. The increase in the concentration of CROTONALDEHYDE as strong lacrimator, control 40% reported bitterness, degrades a sample of alcohol on oxidation. The content of vinylalcohol and benzaldehyde - glycoside with the smell of bitter almonds, exceeds the threshold concentration in substrates, but using a culture of S. cerevisiae In - 855 they are less at 19.5% and 32.9%, respectively. Obviously, the positive results of the studies is due to biochemical changes in the cell as a result of adaptation and selection to the appropriate culture conditions.

Thus, the use of a strain of yeast S.cerevisiae In - 3855 capable brivati molasses wort concentration of carbohydrates 21.0 g/100 cm3will allow to intensify the process of obtaining alcohol to 12.4%, reduce by 30% the formation of metabolites.

Bibliography

1. Marynchenko, VA, Matugas D., Svec NR. The technology of alcohol from molasses // Publishing Association "Vyscha SHKOLA, Kiev. 1975. P.75-153.

2. Mikulov A.N., Sobolev AM, boyar V.M. Fermentation ability of RAS yeast // Izvestiya vuzov. Pish. technology. 1995. No. 5, 6. P.14-16.

3. RF patent №2329302 Method of fermentation of molasses wort / Saarow, Aagalla // B. I. 2008. No. 20.

4. As the USSR №1284998 Strain of the yeast Saccharomyces cerevisiae Y-503 used in the manufacture of bakery products / Saarow, Scicence, Bigeldinova, Atimes // B. I. 1986, No. 3.

5. Kudryavtsev I. Systematics of yeasts. M: in ASSR, 212 S.

Table No. 2
CultureEnzymes(U/mg)
β-fructifera notedataAldolaseProvacteur boxylaseAlcoholiday the rogenaza deficit
S.cerevisiae Y-503 (source)29.3±1.960.33±0.0315.2±1.190.55±0.05
S.cerevisiae In - 3855 (experience)33.4±1.590.38±0.0218.1±1.300.74±0.06
S.cerevisiae I (control)27.4±1.780.26±0.0212.3±1.150.45±0.03

Table 3
The product of fermentationUnitMolasses culture medium with geothermal water
The strain Saccharomyces cerevisiae In - 3855The Saccharomyces cerevisiae strain Y-503
1. Acetaldehydemg/DM32318.744637.48
2. And Eton =At 55.9578.98
3. The ethyl acetate=26.9554.67
4. Propanol-1=3700.845253.26
5. Isobutanol=1116.521299.47
6. Butanol-1=100.42122.44
7. Isoamyl=3593.684244.99
8. Hexanol=6.7827.04
9. CROTONALDEHYDE=27.2868.47
10. Vinylalcohol=580.33720.88
11. Benzaldehyde=62.8193.62
The amount of impurity compounds=11590.316601.3

The strain of the yeast Saccharomyces cerevisiae VKPM B - 3855 used to obtain alcohol.



 

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