RU2394098C2 - Method for cultivation of yeast for alcohol production - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method includes preparation of inoculum and stage of fermentation. For preparation of inoculum, yeast adaptation is carried out to stress agent under aerobic or microaerophilic conditions with illumination by visible light of 40-90 MW/l by means of periodical reinoculation. Fermentation is carried out with application of prepared inoculum and at the level of illumination of 40-90 MW/l. Yield of yeast biomass makes 2.2-4.2 g/l.

EFFECT: method provides for high yield of yeast biomass.

2 tbl, 15 ex

Description

The invention relates to methods for culturing yeast and obtaining the metabolite synthesized by them - ethanol.

A known method of cultivating yeast for alcohol production [1]. The proposed method aims to obtain a pure yeast culture with a high concentration of yeast cells and high fermentation activity. Cultivation is carried out under aerobic conditions on a sterile nutrient medium with a sugar content of 4-6%, as a nutrient medium, the product is used for decantation of post-alcohol stillage stillage and must or sucrose until the concentration of yeast cells in the culture fluid at each stage of cultivation reaches 500-1000 million / ml, after whereby the culture fluid of the last stage is kept under anaerobic conditions for 2-3 hours, during the cultivation, they are fed with sterile wort or a solution of sucrose in the post-alcohol decantation product dard bards to a sugar content in the nutrient medium of 4-6%.

There is also known a method [2] for conducting a fermentation process in which the yeast biomass is prepared in a chemostatic aerobic process, achieving a high cell concentration of 19 g / L.

The biomass prepared according to these options is used in the anaerobic process.

The disadvantages of the methods is an extensive mainly approach to the preparation of seed for the anaerobic process, associated with an increase in the concentration of yeast cells, detailed physiological indicators of the anaerobic process are not given, due attention is not paid to maintaining high physiological activity of cells throughout the anaerobic process, anaerobic processes are carried out with high concentration of biomass, although their specific fermentation activity is small.

Closest to the proposed technical essence is a method for producing yeast biomass, which can be used to prepare seeds of the anaerobic process, including culturing yeast on a carbohydrate-containing substrate in the presence of hydrogen peroxide with preliminary adaptation of yeast cells to hydrogen peroxide [3]. It is noted that when microorganisms previously adapted to hydrogen peroxide are used in microbiological processes, fermentation activity increases (up to 10-20%) in terms of fermentation rate.

The disadvantage of this method is the lack of reproducibility of the positive effect and ignoring the aeration regimes in the preparation of seed.

The objective of the invention is to obtain biomass of yeast with reproducibly high physiological activity in order to intensify the fermentation process and maintaining high physiological activity of the culture during the anaerobic process.

The problem is solved in that for the preparation of seed material, yeast for alcohol production is adapted to a stress agent under aerobic or microaerophilic conditions by illuminating the suspension of cultivated yeast with visible light with an intensity of at least 40 mW / l, by periodic reseeding of yeast culture with exposure to a stress agent, and the subsequent stage of fermentation when inoculated with prepared seed is carried out at a lighting level of at least 40 mW / l.

In the present invention, yeast was cultured in flasks on a rocking chair and in a bioreactor.

The objects of experiments were yeast Saccharomyces cerevisiae strains SL-100 and Meyen race T 985. During cultivation, the optimum temperature for the growth of S. cerevisiae yeast is 28-30 ° C, pH from 3.0 to 6.0.

Experiments with yeast in 250 ml flasks with a medium volume of 50 ml were carried out on a thermostatically controlled shaker with a speed of 220 rpm with an initial pH of 5.5. The components of the medium were sterilized at 0.5 atm in an autoclave for 30 minutes. The seed was 10 vol.%, H ₂ O ₂ (perhydrol) was introduced into various growth phases in an amount of 0.15-1.2 g / l. In a number of experiments, instead of H ₂ O ₂ , irradiation of the yeast with soft ultraviolet was used - 100-400 J / l for 5 sec. The cultivation time was 24-28 hours.

The composition of the medium for the cultivation of S. cerevisiae yeast, g / l: (NH ₄ ) ₂ SO ₄ - 5.0, KH ₂ PO ₄ - 1.0, KCl - 0.15, MgSO ₄ · 7H ₂ O - 0.2 g / l, CaCl ₂ - 0.05 g / l, yeast extract - 0.5, sucrose - 30 or 150, water - tap, pH 5.8.

The experiments in a laboratory bioreactor with a glass shell and a working volume of 3 L were carried out under deep cultivation under sterile conditions with automatic control of pH, pO ₂ , T. The components of the medium were sterilized at 0.5 atm in an autoclave for 30 minutes. Sowing material was 10 vol.%. Soft UV was irradiated through an external circuit with a quartz cuvette.

The flasks and the bioreactor were illuminated with fluorescent lamps, the illumination intensity was determined using a light meter - a TKA-01/3 UV radiometer. In cultivation variants without illumination, the contents of the flasks or bioreactor were shielded from visible light by a opaque material. In flasks, by conducting sequential passages (transfers) under aseptic conditions, depending on the studied factors, yeast lines were obtained under various lighting conditions and without lighting, under aerobic, microaerophilic or anaerobic conditions, when various concentrations of H ₂ O _{2 were} introduced into the culture medium, with different the content of yeast biomass and substrate (sucrose) in the fermentation medium.

In preliminary experiments on flasks in various modes at an initial substrate concentration of 30 g / l, the doses of peroxide application were determined - 0.3-0.6 g / l and exposure to soft ultraviolet radiation of 200-250 J / l for 5 sec, with yeast content cells in the culture medium of 2.2-2.4 g / l, residual substrate concentration <5 g / l, while there was no complete death of the culture. With the effect of the sublethal doses of stress exposure found, a series of passages were conducted.

The parameters found above are not determinative. So, for another strain of S. cerevisiae, a different dose of H ₂ O ₂ or soft UV is needed, at a different concentration of biomass, cultivation can occur at a different initial concentration of the substrate. Finding sublethal doses of stressful effects is individual for each yeast culture. The general determining parameters are the modes of aeration and lighting during the processes.

The following examples illustrate the invention.

Example 1

Seed preparation

Conducting passages (transfers) in flasks when applying peroxide of 0.6 g / l or exposure to soft UV 250 J / l for 5 sec, when the content of yeast cells in the culture medium is 2.2-2.4 g / l when illuminated with visible light intensity 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l, 1000 mW / l in aerobic mode.

Example 2

Seed preparation

Conducting passages (transfers) in flasks when applying peroxide of 0.6 g / l or exposure to soft UV 250 J / l for 5 sec, when the content of yeast cells in the culture medium is 2.2-2.4 g / l when illuminated with visible light intensity of 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l, 1000 mW / l in microaerophilic mode.

Example 3

Seed preparation

Conducting passages (reseeding) in flasks when applying peroxide of 0.6 g / l or exposure to soft UV 250 J / l for 5 sec., When the content of yeast cells in the culture medium is 2.2-2.4 g / l under visible lighting light intensity of 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l, 1000 mW / l in anaerobic mode.

Examples 4-6

Seed preparation

The same as in examples 1-3, but without lighting.

Examples 7-9

Seed preparation

The same as in examples 1-3, but when illuminated with visible light with an intensity of 30 mW / L. A comparison of the fraction of dead cells of different variants of passage (reseeding) with the control variant without exposure to a stress agent at the end of cultivation is given in Table 1. Control variants were carried out for each example, and the results were identical, therefore, the control variant in table 1 is presented in one line.

Table 1 N example 1st passage 5th passage Passage 10 one 60-70% 30-35% 25-30% 2 60-65% 30-35% 25-30% 3 85-90% 90-95% 90-95% four 85-90% 70-75% 70-75% 5 85-90% 70-74% 70-75% 6 90-95% 95-100% 95-100% 7 70-75% 60-65% 55-60% 8 65-70% 55-60% 50-65% 9 85-90% 90-95% 90-95% The control 30-35% 30-35% 30-35%

It is seen that under anaerobic conditions, the cells do not adapt to the stressor (examples 3, 6, 9).

The proportion of dead cells exposed to peroxide or soft UV under aerobic or microaerophilic conditions, when subsequently in the dark, is more than 85%, respectively, for the 1st passage. Without lighting, the proportion of the dead remains high and by the 10th passage about 75% (examples 4, 5).

When the medium is illuminated, the proportion of dead cells drops to the level in the control (without exposure to the stress factor) by the 5th passage for aerobic and microaerophilic conditions (examples 1,2). At the same time, an increase in the intensity of illumination above 40 mW / l does not significantly affect the survival rate and speed of cell adaptation. Thus, scattered light with an intensity of 40 mW / l is enough to restore cell viability. In this case, light acts as an antistressor that helps yeast cells overcome the effects of oxidative stress, probably through the photoreparation mechanism. A decrease in the level of illumination with visible light below 40 mW / L leads to an increase in the proportion of dead cells (examples 7.8 compared with examples 1, 2). In examples 1, 2, there is also a clear correlation between a decrease in the proportion of dead cells, a decrease in the lag phase, an increase in the specific growth rate and resistance to pH (with the acquisition of resistance to H ₂ O ₂ or soft UV cells become resistant to lower pH) as reseeding in lines with stressful effects, i.e. adaptation of the population to the stressor is observed with a change in the cultivation indicators in a favorable direction, while the resistance of cells to the introduction of increased doses of H ₂ O ₂ or exposure to mild UV is also increased: adapted cells withstand 1.8–2.4 g / l of peroxide, whereas in the control variant, the cells die completely when 1.2 g / l of peroxide is added.

The experiments in the reactor with the lines of examples 1 or 2 showed a decrease in the proportion of dead cells in the middle of the exponential phase from 8-10% in the control, to 4-5% in the 5th and 10th passages (transfers), the lag phase is already towards The 5th passage decreased by about 2 times. The addition of hydrogen peroxide leads to the lysis of cells in the control version: at the time of the end of the experiment after exposure to a stressor, the proportion of dead cells reaches 65-70%, the biomass level drops by 35-40% compared with the same version without adding peroxide. When culturing adapted yeasts, there is even a slight increase in the biomass yield after exposure to the stressor. By the end of the experiment, after application, for example, of hydrogen peroxide during cultivation of yeast of the 5th passage, the proportion of dead cells is 30-35%, and of the 10th passage, 20-25%.

Example 10

Seed preparation

As in the cultivation in flasks, when growing yeast lines adapted to peroxide or soft UV in the reactor, for the appearance of physiologically favorable changes, it is important to illuminate the contents of the fermentation medium with visible light with an intensity of at least 40 mW / L. In the dark, after applying H ₂ O _{2 of} 0.6 g / l or exposure to soft UV 250 J / l for 5 s, the proportion of dead cells was 70-75% by the end of cultivation, and 30-35% in the light, which occurred during cultivation adapted yeast of the 5th and 10th passages of examples 1,2 under aerobic or microaerophilic conditions.

The seeds of examples 1.2 after adaptation to peroxide or soft UV when exposed to visible light of at least 40 mW / l can be used as seed for anaerobic cultivation in a bioreactor.

All variants of anaerobic cultivation in the bioreactor were carried out at a substrate concentration (sucrose) of 150 g / l. Sowing material was 10 vol.%.

Example 11

Anaerobic cultivation

Anaerobic cultivation when using non-adapted yeast as an inoculum (control version) when illuminated with an intensity of 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l , 1000 mW / l and without it.

Example 12

Anaerobic cultivation

Anaerobic cultivation using yeast of the 5th passage of examples 1.2 as an inoculum under illumination of intensity 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l, 1000 mW / l.

Example 13

Anaerobic cultivation

Anaerobic cultivation using the yeast of passage 10 of Example 1.2 as an inoculum under illumination of intensity 40 mW / l, 90 mW / l, 150 mW / l, 220 mW / l, 400 mW / l, 500 mW / l, 700 mW / l, 1000 mW / l.

Example 14

Anaerobic cultivation

Anaerobic cultivation without lighting when using the yeast of Example 10 as an inoculum (after applying H202 in aerobic or microaerophilic modes and culturing in the dark).

Example 15

Anaerobic cultivation

Anaerobic cultivation using yeast prepared as an inoculum prepared according to the prototype under conditions of changing the illumination of the laboratory room with natural visible light when day and night change.

table 2 N example Specific growth rate, h ^-1 % dead in the middle of the exp. phase % dead at the end of the experiment The highest fermentation activity (ml CO ₂ / l · h) Fermentation time, h The yield of alcohol,% The biomass yield, g / l eleven 0,046 15-20% 75% 680-700 73 6.2-6.4 four (the control) 12 0.059 10-12% 60-65% 880-900 57 6.2-6.4 4.1 13 0,067 8-10% 60-65% 980-1000 52 6.2-6.4 4.2 fourteen 0,044 40-45% 90-95% 480-500 93 4.0-4.1 2.2 fifteen 0.05-0.06 10-15% 65-70% 830-850 59 6.2-6.4 4.1

The study of the role of lighting and aeration in the preparation of seed and lighting during anaerobic cultivation allowed us to offer a new version of the process of alcoholic fermentation compared to the prototype (example 15), in which the above parameters were not taken into account when preparing the seed.

Thus, the possibility of a significant improvement in the growth rates of yeast cultures and the process of alcoholic fermentation with the combined exposure to yeast with doses of stress factors (hydrogen peroxide or soft ultraviolet radiation) and an anti-stress factor, in particular visible light, has been shown, which allows us to propose a new method for conducting the process of alcoholic fermentation. According to the proposed method, in order to maintain high physiological activity of saccharomycete yeast, seed is obtained during yeast growth under aerobic or microaerophilic conditions and combined exposure to visible light and H ₂ O ₂ or soft ultraviolet of the UVA / UVB range during passivation (transfers) of the culture.

The proposed method allows you to maintain high physiological activity of yeast cells and helps to increase the productivity of the process - reducing fermentation time by 30% compared with the control, and 12% compared with the prototype.

Literature

1. RF patent N 2136746 A method of culturing yeast for alcohol production, priority 17.08.1998.

2. Patent WO 2007038833 ETHANOL FERMENTATION PROCESS AND PRODUCTS, priority 03.10.2005.

3. RF patent N 2268924 A method for producing yeast biomass, priority 23.11.2004.

Claims (1)

A method of cultivating yeast for alcohol production, including the preparation of seed and a fermentation stage, characterized in that for the preparation of seed the yeast is adapted to a stress agent under aerobic or microaerophilic conditions with visible light level of 40-90 mW / l by periodic reseeding of yeast culture with exposure to a stress agent, and the subsequent stage of fermentation when inoculated with prepared seed is carried out at a lighting level of 40-90 mW / l.