SU1117319A1 - Bp saccharomyces cerevisae paca heat tolerant yeast strain used for brewing starch-containing raw material in production of ethyl alcohol and method of brewing starch-containing raw material in production of ethyl alcohol - Google Patents

Abstract

1. Thermotolerant strain of Saccharorayces cerevisiae yeast race VP (collection of industrial cultures of All-Union Scientific Research Institute of Genetics and Selection of Microorganisms, collection number CMPM U-408), used to ferment starch-containing raw materials in the production of ethyl alcohol. 2. A method of fermenting starch-containing raw materials in the production of ethyl alcohol, which involves preparing a seed culture, preparing a nutrient medium by mixing starch-containing raw material with the salts of carbamide and diammonium phosphate necessary for the yeast, yeast generation, saccharification and transferring the yeast culture from the wort to 17 a head-mounted battery fermenter with sequential movement of a continuous flow method and the inflow of saccharified wort (L characterized in that, with the aim of Ensuring the sterility of the process, speeding up the fermentation process, by maintaining a constant optimum for breeding yeast ratios of nitrogen to fermentable water, use the thermotolerant strain of the Saccharomyces cerevisiae yeast race HP as a seed culture; m fermentation by three-stage subculture of it on the saccharine wort with a concentration of 17-19% solids with a gradual increase in temperature from 22 to 28 s, from 28 to 30 to 32 C, with this urea separated into two parts in a ratio of 1: 4, most of which is introduced into the saccharified mash before the first passaging step, and smaller - on Lag-phase fermentation process together with the inflow saccharified mash.

Description

The invention relates to the alcohol industry and is a thermotolerant strain of the Saccharomyces cerevisiae yeast race VP for fermenting starch-containing raw materials in the alcohol industry and a method for fermenting starch-containing raw materials.

A known thermotolerant strain of yeast Saccharomyces cerevisiae of races K-81 is used to serve starch-containing raw materials in the production of alcohol l}.

The disadvantage of this strain is its inability to ferment highly concentrated wort (17-19% DM).

The closest in technical essence to the present invention is the Saccharomyces cerevisiae strain CP, which ferments a highly concentrated wort 2.

A disadvantage of the known strain is that it is not thermotolerant and already at a temperature above 32. Autolysis occurs, which leads to the infection of the environment by extraneous micro organisms and to the loss of alcohol.

Methods are known for the fermentation of starch-containing raw materials in the production of alcohol, involving the introduction of yeast and the fermentation of grain-carto-wort by the continuous-flow method 3j.

The closest technical solution to the present invention is a method of fermenting starch-containing raw materials in the production of ethyl alcohol, which involves preparing a seed culture, preparing a nutrient medium by mixing the starch-containing raw material with the yeast salts, urea and diammonium phosphate, yeast generation and saccharification, and yeast culture. 1719% CB in the head battery fermenter with sequential movement in a continuous flow method and flow of oil go wort 4j.

The disadvantage of the known method is that the fermentation process takes place at a temperature not above, with an increase in temperature, a significant autolysis of the yeast cells and infection of the medium occurs. Since the optimal balanced nutrition of nitrogen and carbohydrates is impaired in the brocade process, the yeast loses its activity, which, combined with the infection of the medium, leads to a significant loss in alcohol output. After 48 hours of digestion, there are no reserves to lengthen the process and to obtain an additional alcohol yield. There comes the infection of the medium and the loss of fermentation activity by the yeast.

The aim of the invention is to obtain a thermotolerant yeast strain capable of fermenting highly concentrated wort at elevated temperatures, ensuring sterility of the process, accelerating the fermentation process by maintaining a constant ratio of nitrogen to fermented carbohydrates for yeast reproduction.

The proposed strain was isolated from production yeast and adapted to high concentrations of dry substances in the wort.

Selection was carried out in two stages. The first stage consisted in separating the yeast race from production capable of fermenting raw materials at elevated temperatures (34-36 ° C). To do this, a yeast culture was applied at the plant to be dispersed in the form of colonies on the wort and grown at 34-36 ° C. Colonies differing from the original yeast race were tested for the ability to ferment carbohydrates.

At the second stage of selection, the method of adapting the thermotolerant race to high concentrations of dry substances was applied. A weekly replanting of the thermotolunt race of medium with higher dry matter concentration and sieving of yeast in the form of colonies for wort agar selected a race, a distinctive feature of which was the ability to ferment mash with a high concentration of dry matter (up to 20%) at 34-36 C.

The strain is stored in the collection of the Museum of Industrial Microorganism Cultures of the All-Union Scientific Research Institute of Genetics and Breeding, collection number U-408.

The proposed strain of the yeast Saccharomyces cerevisiae race VP has the following morphological and cultural characteristics.

Morphological signs. Yeast cells are oval or ovoid. Cell size 7.5x5.9 microns Reproduction by budding. The shape of yeast cells grown on wortylocene for 26-28 hours at 32-34 ° C, ovoid, oval, cells of elongated shape are rarely found.

Giant colonies 10-day j culture on wort agar. Colony shape: round. Colony size: 26-28 mm in diameter. The nature of the edge: large-blade with a radial isch. JO Surface: dim. In the center of the colony there is a convex roller of irregular shape with a wrinkled surface.

Colony color: white with creamy red. Colony profile: bulging in the center.

Barcode on wort agar. Growth: powerful, tight.

Nature of the Kra: small-scale. 20 Profile: bulging. Color, stroke - dull.

Growth care in wort agar. Growth along the length of the injection, thickening upwards. .25

Injection form: hilly. No dilution was observed. The nutrient medium is permeated with gas bubbles.

Physiological signs. zo Optimum growth temperature 34/36 C.

Relation to carbon sources. Absorbs glucose, galactose, sucrose, maltose. Weakly assimilates xylose, arabinose. Does not digest small, dextrins, melibiosis.

Relation to nitrogen sources. Well assimilates urea, peptone, yeast autolysate, ammonium nitrate and ammonium chloride, potassium nitrate, calcium, sodium 1). Does not consume ammonium sulfate. Optional anaerobic.

In order to achieve the goal of fermentation of starch containing raw materials in the production of ethyl alcohol, which involves preparing a seed culture, preparing a nutrient medium by mixing the starch containing raw materials with yeast salts of urea and diammonium phosphate, yeast production, saccharification, donation, charification, cooking, and sugar. and wort with a concentration of 17–19% of dry substances in the head fermenter of the battery with sequential movement by the continuous flow method and the inflow of saccharified with As a seed culture, a thermotolerant strain of the Saccharomyfees cerevisiae yeast race VP was used as a seed culture, and during the yeast generation, this strain was adapted to ulo. VIYAM fermentation by three-stage transferring it to a saccharified mash with a concentration of 17-19% solids with a gradual increase in temperature from 22 to 28 ° C, from 28 to 30 ° C and from 30 to 32 ° C, while urea is divided into two parts in the ratio 1: 4, the larger of which is introduced into the saccharified wort before the first step of seeding, and the smaller - into the lag phase of the fermentation process, together with the influx of saccharified wort.

The method is carried out as follows.

The Saccharomyces cerevisiae rasa VP culture was grown on wort agar in test tubes, stored culture, then subcultured on fresh nutrient medium, wort agar.

Then, a pure culture is diluted by successively reseeding.

The obtained sowing yeast is adapted to the higher fermentation temperature, carried out successively subcultured in three stages on saccharified, highly concentrated tycno with a gradual increase in temperature from 22 to 28 ° C, from 28 to and from 30 to 32 ° C.

Urea is divided into two parts in the ratio of 1: 4 and introduced respectively into the saccharified wort before the first stage of adaptation and into the lagfaz during the fermentation process, together with the influx of yeast and saccharified wort.

The ratio of assimilable nitrogen to the fermented carbohydrates during the whole fermentation process is maintained at 1: 200.

Fermentation lead continuous flow method.

; v

Example 1. Culture Sacch. The erevisiae race of VP is infused on a conditional agar containing 7% of CB in robirka at 32 ° C for 24 hours. It is cultured for 5 months. After the specified rock has elapsed, in order to preserve the fermentation activity, the culture is sown on a suitable nutrient medium, wort agar.

511

Dilution of the pure culture is then carried out by successive transfer from the culture tube into a flask containing 0.5 liters of sterile wort. The wort is prepared from crushed malt (0.7 l of water is consumed per 1 kg of malt), saccharified at 58 ° C for 2 hours. Its concentration should be 12 ° according to the sugar meter,

The flask with the wort and yeast is placed in a thermostat at, after 20 h, the fermented yeast wort is poured, into the bottle with 5 liters of sterile same wort, acidified to 0.8 d. The bottle with the wort is placed in a 30 C thermostat with a tap equal to 5 according to the sugar meter, the contents are transferred to the mother liquor from 5 gave the mash with a concentration of 10 according to the sugar meter, acidified to OjE,

To dilute the yeast in the mother liquor, an unfiltered wort prepared as follows is used.

For preparation of 1 yarn, 1.3 kg of rye is consumed of yeast wort. flour and 1.9 kg of chopped green malt. Flour mixed with water at 30 ° C. The mixture was kept for one hour, cooled to 60 ° C, after which crushed malt was poured. The grating is continued for 1 hour at 58 ° C and 1.5 hours at 65 ° C. Then the mash is held at 80 ° C for 30 minutes, cooled to 50 ° C and acidified to 0.75 ° sulfuric acid. .

The sowing yeast from the bottle to the mother liquor is set in a volume of 10% at 30 C.: From the mother liquor the yeast is transferred for subsequent reproduction into yeast.

Characteristics of mature yeast are presented in table. 1, the dynamics of the accumulation of yeast cells in Table. 2

Example 2 The inoculated yeast obtained according to Example 1 g is adapted to an elevated temperature of fermentation, carried out by reseeding in three stuiieini on a wort prepared from ground wheat. To obtain the wort, the wheat is crushed, mixed with water - gz ratio 1: 2.8 to a concentration of 18% CB, saccharified with a mixture of malts for 2 hours.

In the saccharified wort, diammoiiphosphate is set in a quantity of 150 g and urea in an amount of 560 g per 1 m / wort, mixed, pasteurized for 20 minutes at 80 ° C, acidified to pH 3.6-3.8, cooled to

nineteen

The first stage of adaptation is 22-C, the yeast is sown on this medium in an amount of 10 /: to the volume of saccharified wort and fermented for 18 hours, maintaining the fermentation temperature up to 28 C. Then the mash is again prepared in the same way, the yeast is added in an amount . 10% by volume of the wort and ferment for 16 hours, maintaining the temperature at 28-30 ° C (due to the heat generated during fermentation). Similarly, the third stage of adaptation is carried out for 16 h, maintaining the temperature at 30-32 ° C.

The ratio of nitrogen to carbohydrates is set optimal for yeast, equal to 1: 200, with the introduction of urea at the stage of yeast generation.

From the 3rd stage, the adapted for production conditions the yeast is subcultured into three yeast plants per wort prepared by the above method.

After 14 hours of fermentation, the production yeast from the 3 yeast cells is transferred to the head battery fermenter. Fermentation is carried out in a fermenter with a capacity of 100 m at a wort inflow rate of 12 m per hour and a yeast supply of 30 m together with urea, which is set at 140 g per 1 m of wort to maintain the ratio of nitrogen to fermentable carbohydrates equal, as already proved, 1: 200 The temperature of the supplied wort for fermentation is 30 ° C. Fermentation is carried out by a continuous-flow method for 60 hours at a temperature at the stage of main fermentation up to 38 ° C. The alcohol yield is 65.5 dal of 1 ton of starch.

Technological parameters of a mature beer are presented in table. 3

The implementation of the process is achieved by using a thermotolerant strain of yeast and the introduction of urea in two steps - at the yeast generation stage and at the initial stage of the fermentation process. I

When using a thermotolerant strain, yeast survives at high temperatures. In this case, significant autolysis of the yeast cells does not occur, their fermentation activity is preserved and thus the development of extraneous microflora is inhibited.

This effect, aimed at preserving the vitality and high activity of the yeast, is even more enhanced by the introduction and maintenance of sb1117

lance feed for nitrogen and yivods. Nitrogen is needed to create proteins, a yeast cell, including structural proteins, constituent cells, spore membranes, and 5 structural elements of the cell's internal particles, and to create the entire enzyme system of the cell.

Carbohydrates are necessary for cell respiration, protein biosynthesis in those cases where there are no necessary amino acids.

Yeast from carbamide forms one of the vitamins necessary for their development - biotin.

The maximum possible speed of reproduction of yeast is achieved only with a proper and strict balance of the processes of anabolism and catabolism. The rate of yeast reproduction on 20 rich nutrient media largely depends on a balanced food source.

The intracellular concentration of nitrogenous and carbohydrate compounds increases very significantly during the lag period, so it is important to introduce the nitrogen source in exactly two phases at the stage of active yeast multiplication and during the lag phase when young cells are already. Q consumed part of the nutrients and it needs to be replenished so that the Control

nineteen . The increase in nitrogen to carbohydrate was constant and equal to 1: 200.

After the lag-phase, the viability of the yeast proceeds under constant established conditions, the process of the biosynthesis of alcohol begins, and this ratio remains constant.

If carbamide is given only at the yeast generation stage, Td will lead to an increase in the environment of the nitrogen source and energy in the absence of carbohydrate balance, which adversely affects the physiological state of the cell. The optimum ratio is 1: 200, because, above or below these limits, the energy unused on cell proliferation is dissipated as heat and accumulated in storage and oxidized metabolic products.

The advantages of the proposed technical solution in comparison with what is known are presented in Table. BUT.

Thus, the proposed strain and method allow the highly concentrated wort to be fermented at elevated temperatures, to ensure the sterility of the process, to accelerate the process of fermentation by maintaining a constant ratio of nitrogen to fermentable carbohydrates.

Table 1

Average results 27 22 11.0 19.6 28.5 19.7 0.0 0.8 0.4 0.4

ten

1117319 Continuation of the table. one

13

The process proceeds at a temperature not higher. The increase in temperature leads to infection, since the yeast is not thermotolerant and undergoes autolysis. Note. since there is no increase in output.

14 Table

1117319

64.7 dal / t

48 hours of starch A further increase in fermentation time is not possible, acidity and loss of alcohol. No reserves to increase

Claims (2)

1. Thermo-tolerant strain of yeast Saccharomyces cerevisiae race VP (culture collection of industrial microorganisms of the All-Union Scientific Research Institute of Genetics and Breeding of Microorganisms, collection number CMPM U-408) used for fermentation of starch-containing raw materials in the production of ethyl alcohol. 2. A method of fermenting starch-containing raw materials in the production of ethyl alcohol, which involves preparing a seed crop, preparing a nutrient medium by mixing starch-containing raw materials with salts necessary for yeast - carbamide and diammonium phosphate, yeast, irration, saccharification and transfer of yeast culture from wort with a concentration of 17-19% dry matter in, the head fermenter of the battery with successive continuous flow method and inflow of sugared wort, characterized in that, for the purpose of providing To ensure sterility of the process, accelerate the fermentation process by maintaining a constant on-to-optimal ratio of nitrogen to fermentable carbohydrates for yeast reproduction, a heat-tolerant strain of the Saccharomyces cerevisiae strain of the VP race is used as a seed culture; during yeast generation, this strain is adapted to the fermentation conditions by three stepwise reseeding it on saccharified wort with a concentration of 17-19% solids> with a gradual increase in temperature from 22 to 28 ° С, from 28 to ЗО ^ С, from 30 to 32 С, while urea is divided are poured into two parts in a ratio of 1: 4, the larger of which is introduced into the saccharified wort before the first stage of reseeding, and the smaller into the Lag phase of the fermentation process together with P17319 lump of sugared wort.