RU2361919C1 - Method of obtaining biocatalyst for alcoholic fermentation - Google Patents

Abstract

FIELD: food products; alcoholic beverages. ^ SUBSTANCE: method of obtaining biocatalyst for alcoholic fermentation comprises increasing biomass of yeast Saccharomyces cerevisiae and immobilising it by including it in helium matrix by way of mixing with gelling material solution with its subsequent curing by ions Ca++. Gelling material is represented by mixture of exopolysaccharide with molecular weight 1.5105-2.5105 Da, derived from Paracoccus denitrificans All-Russian collection of industrial microorganisms B-8617 bacteria strain and sodium alginate in mass ratio 0.1-0.9:3.5.-4.5. ^ EFFECT: obtaining highly active biocatalyst with long work period for alcoholic fermentation basing on free yeast cells for industrial production of ethanol as chemical while using it as fuel. ^ 1 tbl, 2 ex

Description

The invention relates to the biochemical industry, and in particular to a method for producing immobilized yeast-based biocatalysts for alcoholic fermentation to produce ethyl alcohol as a chemical product used, in particular, as fuel.

Free yeast cells are the traditional biocatalyst of alcoholic fermentation of malt and grape must, wine materials, fruit, vegetable and other vegetable juices, sugar and starch-containing materials, etc., in order to obtain alcohol-containing compositions.

The need to increase the manufacturability of the fermentation process required the development of new biocatalysts, which, in particular, increased the duration of their use in fermentation processes, simplified the stage of separation of the biocatalyst from the fermentation medium, the possibility of regeneration and reuse of the spent biocatalyst, improved processability of continuous processes of alcoholic digestion.

A wide range of patented technical solutions is known [for example, JP 57-150385, JP 60153794, JP 1067189, US 5079011, US 5334229, DE 3432923, FR 2320349, FR 2359202, FR 2601687, FR 2600673, FR 2586256, EP 388588, FR 2668081, ES 2192986], which includes methods for producing alcoholic fermentation biocatalysts based on yeast that partially solve the problems that arise when using free yeast cells in the production of wines, beer, effervescent drinks with different alcohol contents, and champagne material.

A common essential feature of these known methods is immobilization by incorporation of free yeast cells with a gel carrier matrix to form solid, usually spherical particles, and the curing of the initial gelling material (for example, sodium or potassium salts of alginic acid) is carried out either by ion exchange using divalent ions or trivalent metals (calcium chloride, barium, strontium, aluminum), or by polymerization included in the original yeast suspension monomer, for example acrylamide, or by specific treatment of the initial yeast suspension, which includes a polymer, for example polyvinyl alcohol, with the formation of so-called cryogels.

However, the above technology for producing alcoholic fermentation biocatalysts is intended to produce biocatalysts for the preparation of alcohol-containing drinks, such as wine, beer, champagne, effervescent drinks with different alcohol contents, and, according to the information given in the descriptions of the above patents, cannot provide highly active, preserving for a long time interacting with the fermentation medium, both its biochemical and mechanical properties, biocatalyst, pre designated for commercial production of ethanol.

Closest to the invention in terms of essential features is a method for producing a biocatalyst of alcoholic fermentation, used to produce ethanol, described in patent DE 3432923, characterized by the following set of essential features.

Free yeast cells are suspended in physiological saline and mixed with a pre-prepared sterile aqueous solution of sodium alginate. The resulting suspension of a solution of sodium alginate with yeast cells is served in the form of drops in a bath with a solution of calcium chloride. When droplets enter the bath as a result of the exchange of Na ⁺ ions for Ca ⁺⁺ ions, 3 mm gel-like granules (pearls) are formed. The gel granules that have arisen through ionotropic formation are kept in a solution of calcium chloride with constant stirring for 30 minutes, sieved and washed repeatedly with running water. Then washed granules for 15 minutes with constant stirring, rinse in a solution of sodium alginate. Due to the diffusion of Ca ⁺⁺ ions from gel pearls into a solution of sodium alginate, a rather thick shell of Ca alginate is formed around the pearl granules. The granules are again washed with running water and immersed for 1 hour in a solution of calcium chloride, after exposure to a solution of calcium chloride, the granules are again subjected to repeated washing with running water. The obtained biocatalyst granules are sterilized by irradiation for 5 minutes with a 6 W UV lamp.

A disadvantage of the described known method for producing an alcoholic fermentation biocatalyst is the complexity of its preparation on an industrial scale with the high speed and low cost of producing a significant amount of high-quality biocatalyst, in particular because of its multi-stage nature, as well as the difficulty of preventing agglomeration of a liquid containing a crosslinking agent on the surface agent (CaCl ₂ ), gel particles with free yeast cells included.

In addition, according to the known method, a biocatalyst is obtained, the particles of which are surrounded by a "sufficiently thick layer" of Ca-alginate gel, additionally cured with Ca ⁺⁺ ions, which does not contain immobilized free yeast cells. This layer provides a longer catalyst life, making it difficult for yeast cells to exit the matrix of the gel carrier, but making it harder to mass transfer between the liquid phase of the nutrient medium and the gel phase of the catalyst, which contains, in fact, the active biocomponent itself - yeast cells, which significantly reduces the activity of the biocatalyst in whole.

The objective of the invention is to develop a simpler, more technologically advanced method for producing a highly active alcohol-fermentation biocatalyst with an increased shelf life based on free yeast cells (immobilized granular yeast (hereinafter - IHD) for the industrial production of ethyl alcohol as a chemical product used as fuel for due to the exclusion of agglomeration of gel particles with included free yeast cells.

The problem is solved in that in the method for producing a biocatalyst of alcoholic fermentation, including the growth of biomass of yeast and its immobilization by incorporation into a gel matrix by mixing with a solution of a gel-forming material followed by its curing with Ca ⁺⁺ ions, according to the invention, a mixture of exopolysaccharide with molecular is used as a gel-forming material weighing 1.5 · 10 ⁵ -2.5 · 10 ⁵ Da obtained from the bacterial strain Paracoccus denitrificans VKPM B-8617, and sodium alginate in a mass ratio of 0.1-0.9: 3.5-4.5.

The exopolysaccharide used is obtained from the bacterial strain Paracoccus denitrificans VKPM B-8617 (RU 2006127257, 2006).

The strain of Paracoccus denitrificans VKPM B-8617 is characterized by the following features. In the active phase, Paracoccus denitrificans VKPM B-8617 cells are thick short bacilli 1-1.5-1.5-2.0 μm in size. Bacterial cells form opal colonies. There is a need for additional growth factors (yeast extract and pantothenic acid), growing on media containing methanol. The strain of Paracoccus denitrificans VKPM B-8617 is not pathogenic for mice.

The specified strain propagates at 20-29 ° C; stored at + 4 ° C on beveled glucose-potato agar or wort agar. The medium is sterilized at 0.75 atm and a temperature of 110 ° C. The frequency of reseeding -1 time in 4-5 months.

Cultural and morphological characteristics of the strain of Paracoccus denitrificans VKPM B-8617.

Bacterial cells are gram-negative, motionless. In the logarithmic phase, the culture is thick short sticks, in the stationary one - coccoids arranged in pairs, less often in short chains. They do not form a dispute. Cell reproduction is carried out by binary division.

When growing on wort agar or on mowed glucose-potato agar, the strain forms convex shiny mucous opal colonies of regular shape, 4-6 mm in diameter. When growing on a liquid medium with ethanol, as the only carbon source, it forms a highly viscous viscous suspension of a light cream color. When repeated screening on a solid rich sugar-containing medium, strain dissociation was not detected. Stable is the mucoid form of bacterial cells, which synthesizes a stable product with a stable yield. Colonial-morphological variability is the uniformity of cells.

Physiological and biochemical characteristics.

Aerobe. Catalase-positive, oxidase-negative, acid-unstable. There is superoxide dismutase, peroxidase is absent. The temperature range of growth is 10-42 ° C. The optimum growth temperature is 24-30 ° C, pH 5.5-8.0, optimum pH 7.0. It does not dilute gelatin. Starch does not hydrolyze. Milk does not ferment. It does not restore nitrates, does not form hydrogen sulfide, indole and acetoin.

The strain of Paracoccus denitrificans VKPM B-8617 refers to microorganisms that are not pathogenic for humans, according to the classification of microorganisms given in the Sanitary rules SP 1.2.731-99.

The strain is cultivated under aerobic conditions. The optimum pH is 7.0. Grows on media containing carbohydrates. Uses inorganic and organic nitrogen sources. Needs additional growth factors.

The following is a list of the components of the culture medium for the cultivation of Paracoccus denitrificans VKPM B-8617 (wt.%):

KH ₂ PO ₄ · 3H ₂ O 0.1-2 NaCl 0.05-1 NH ₄ NO ₃ · 2H ₂ O 0.1-2 MgSO ₄ · 7H ₂ O 0.01-1 FeSO ₄ · 7H ₂ O 0.0001-0.01 CaCl ₂ · 2H ₂ O 0.005-0.05 Pantothenic acid 0.0001-0.001 Yeast extract 0.001-0.1 Glucose 0.5-5.

Ethanol is used as an additional carbon source in an amount of from 0.5 to 2 vol.% Per 100 vol.% Of the nutrient medium.

It is possible to obtain an exopolysaccharide with identical properties on an alternative nutrient medium, the advantage of which is its cheapness compared to the above.

The following is a list of components of an alternative nutrient medium for the cultivation of Paracoccus denitrificans VKPM B-8617 (wt.%).

KH ₂ PO ₄ · 3H ₂ O 0.1-2 NaCl 0.05-1 NH ₄ NO ₃ · 2H ₂ O 0.01-0.5 MgSO ₄ · 7H ₂ O 0.01-1 CaCl ₂ · 2H ₂ O 0.005-0.1 Water up to 100.

As sources of carbon and growth factors, ethanol in an amount of 1.5 vol.% And whey in an amount of 30 vol.% Were used.

The cultivation of Paracoccus denitrificans VKPM B-8617 to produce exopolysaccharide is carried out in a fermenter at a temperature of 28-40 ° C under conditions of aeration and stirring at a concentration of dissolved oxygen of 20-40%, pH of 7.0, for 24-48 hours. The strain productivity under the conditions described above is optimal and amounts to (10.5 ± 0.8) × 10 ⁸ cells / ml in biomass, and from 6 to 10 g / l in the amount of EPS synthesized.

The exopolysaccharide produced by the bacterial strain Paracoccus denitrificans VKPM B-8617 was isolated from the culture fluid and purified by the method described below. The culture fluid containing EPS was dialyzed against distilled water for 5 days. Then it was diluted with distilled water 3 times and the cells were separated by ultracentrifugation. The supernatant was concentrated in vacuo with slight heating to the initial volume, after which the EPS was precipitated by the addition of isopropanol. The EPS precipitate was washed with pure isopropyl alcohol and dried at a temperature of 40 ° C.

EPS isolated by the described method is a mass of entangled short fibers (up to 15 mm) from light yellow to white with a moisture content of 10-12% and a polysaccharide content of 95-99%.

The exopolysaccharide is formed by glucose, galactose, mannose, rhamnose residues (5: 2: 4: 1, respectively), includes glucuronic and pyruvic acids, as well as acyl groups. The molecular mass of exopolysaccharide, determined by gel filtration on a Sephadex G-200 (Sweden), lies in 0.5 × 10 ⁶ -2 × 10 ⁷ D.

The exopolysaccharide is soluble in water and polar organic solvents such as dimethylformamide and dimethyl sulfoxide, forming highly viscous true solutions. Aqueous solutions of exopolysaccharide have pseudoplasticity and thixotropy. With an increase in the concentration of an aqueous solution of exopolysaccharide to 0.1%, its kinematic viscosity sharply increases.

EPS is precipitated from aqueous solutions by alcohols (e.g., methanol, ethanol, isopropanol) and ketones, e.g., acetone. The addition of inorganic salts (CaCl ₂ ) to the EPS aqueous solution results in the isolation or so-called salting out of EPS.

A characteristic property of exopolysaccharide is its ability to structure not only aqueous, but also hydrocarbon-containing systems. When water solutions of EPS (0.2-0.7%) are mixed with normal hydrocarbons (C ₆ -C ₁₂ ), for example, with a volume ratio of 1: 9, respectively, a stable, non-time-stratifying emulsion is formed, characterized by pseudoplasticity and thixotropy.

The dynamic viscosity of such emulsions at a shear rate of 3 s ^-1 is 2500-4000 MPa · s.

An important property of the polysaccharide polysaccharide produced by the bacterial strain Paracoccus denitrificans VKPM B-8617 that is important for use in a number of production areas is the independence of the dynamic viscosity (at low shear rates) of its aqueous solutions from the temperature of the solution up to 90 ° C. The rheological properties of aqueous solutions remain practically unchanged even with their repeated preliminary heating up to 130 ° С followed by cooling to the initial temperature (usually 20 ° С).

The remarkable properties of this polysaccharide are also its film-forming and fiber-forming properties.

The unique structuring properties of this exopolysaccharide and its chemical composition make it possible to pre-form such a structure of a polysaccharide solution (due to the high viscosity of the initial solution of exopolysaccharide at a low concentration) when it is mixed with an aqueous solution of sodium alginate to form the subsequent structure of the inclusion matrix as a result of ionotropic exchange with ions of the curing agent, which ensures the absence of the agglomeration process formed on the surface of the curing the solution to the particles of the biocatalyst. In addition, during the operation of the biocatalyst (IHD) in the process of alcoholic digestion, the physical structure and chemical composition of the cured gel matrix provide it with the ability to retain free yeast cells immobilized by it for a long time, preventing their leaching during interaction with the fermentation medium, and facilitate this mass transfer between the liquid phase of the nutrient medium and the gel phase of the particles of the biocatalyst.

The invention consists in the following.

To build biomass, the cultivation of yeast, for example, of the genus Saccharomyces cerevisiae, is carried out in fermenters under conditions of aeration on a nutrient medium, including: glucose 15-20 g / l; (NH ₄ ) ₂ SO ₄ 15-18 g / l; MgSO ₄ · 7H ₂ O - 4-5 g / l; To ₂ NRA ₄ 5-6 g / l; yeast extract - 1-1.5 g / l; water - up to 1 liter. The optimum temperature for growth is maintained in the range of 28-32 ° C, the optimal pH is from 3.5 to 6.5. The cultivation time is 36-48 hours. The result of the cultivation of these bacteria is to obtain biomass in the form of a suspension of free yeast cells, which is further used in the method for producing alcoholic fermentation biocatalyst according to the invention (IHD).

Powdered sodium alginate is introduced into a 0.1-0.9% (wt.) Aqueous solution of exopolysaccharide in an amount providing a concentration of sodium alginate in a solution of polysaccharides of 3.5-4.5% (wt.), The dynamic viscosity of the solution is 500- 1000 cp. The resulting solution is sterilized by autoclaving it for 18-22 minutes at a temperature of 118-124 ° C, then it is cooled to room temperature.

Equal volumes of the obtained sterile polysaccharide solution and suspension of free yeast cells are mixed. The resulting suspension in the form of droplets is pumped through a calibrated capillary with a diameter of 0.5 to 5 mm from a height of 10-30 cm (vertically) into a solution of a crosslinking agent - a 2-2.5% aqueous solution of calcium chloride, taken in excess . As a result of the interaction of droplets of a suspension of a solution of polysaccharides and free yeast cells with a solution of calcium chloride as a result of a surface exchange of sodium ions of sodium alginate and calcium ions from a solution of a crosslinking agent, spherical gel granules are formed with a diameter of 1-4 mm (height 10-30 cm is optimal for the formation of spherical granules). The resulting gel granules are kept in a solution of calcium chloride for 30-40 minutes for their final (approval, then washed with water, previously sterilized by autoclaving for 30 minutes at 118-124 ° C.

The obtained elastic white translucent spherical granules with a diameter of 1-4 mm are a biocatalyst of alcoholic fermentation (IHD), which are used in the fermentation fermentation of sugars to produce ethanol as a chemical product, used in particular as a fuel.

Biocatalyst granules (IHDs) include a gel matrix cured by ionotropic exchange of divalent calcium ions, consisting of a mixture of exopolysaccharide and Na-Ca alginate, and free yeast cells immobilized by a cured gel matrix.

This biocatalyst exhibits a high fermentation activity in continuous fermentation processes and a high conversion of sugars. This biocatalyst can be successfully used both in batch and continuous fermentation processes.

Tests showed that the biocatalyst according to the invention for six months in the processes of alcoholic digestion to obtain ethanol as a chemical product used as fuel, has not undergone significant changes not only in its biocatalytic properties, but also in mechanical characteristics. Moreover, ethanol in its characteristics fully met the requirements of ASTM D 5798-99 Standard Specification for Fuel Ethanol for Automotive Spark-Ignition Engines.

The raw material for the production of ethanol can be various plant materials containing fermentable sugars in sufficient quantities or other saccharinated carbohydrates, which include starch-containing materials traditionally used for this purpose - grain (rye, wheat, corn, barley, oats, millet) and potatoes sugar-containing materials - molasses (a waste of sugar and starch-syrup production), defective sugar beets, as well as wood and agricultural waste.

The use of an exopolysaccharide with a molecular weight, as well as a mass ratio of the exopolysaccharide and sodium alginate, which go beyond the above values, does not produce the biocatalyst of the desired characteristics.

The following examples illustrate a variant of the claimed invention, but do not limit it.

Example 1

Yeast Saccharomyces cerevisiae SL 100 is grown in flasks under microaerophilic conditions (stationary) on a nutrient medium of the following composition: Glucose - 20 g / l; (NH ₄ ) ₂ SO ₄ - 18 g / l; MgSO ₄ · 7H ₂ O - 4 g / l; K ₂ HPO ₄ - 5 g / l; yeast extract - 1 g / l; at a temperature of 30 ° C, pH 5.5. The cultivation time is 36 hours. The final concentration of dry biomass in 100 ml is 12.3 g. Yeast cells are in an exponential growth phase.

In a 0.1 wt.% Aqueous solution of exopolysaccharide with a molecular weight of 1.5 · 10 ⁵ -2.5 · 10 ⁵ Da, sodium alginate is added in an amount of 4 g per 100 ml of solution (mass ratio 0.5: 4) and stirred until it is completely dissolved. The resulting polysaccharide solution was autoclaved for 20 minutes at a temperature of 121 ° C. Cooled to room temperature, the polysaccharide solution (in the complete absence of air bubbles) is mixed with an equal volume (100 ml) of the cell suspension. The resulting suspension is homogenized and, using a pump, through calibrated capillaries with an inner diameter of 1.5 mm at a speed of 5 ml / h, vertically from a height of 20 cm is fed into a reactor filled with 2 liters of a 2.2% aqueous solution of calcium chloride. The solid spherical granules formed upon contact of the droplets of the suspension with a solution of a curing agent (CaCl ₂ ) are then kept in a solution of calcium chloride for 30 minutes, after which they are washed with sterile water. Biocatalyst (IHD) is a white (sometimes transparent) elastic spherical granules with a diameter of 1-4 mm.

Example 2

The washed granules of the biocatalyst (IHD) obtained in example 1 are loaded into the reactor and 2 l of a sterile nutrient medium of the following composition are added: glucose - 150 g / l; (NH ₄ ) ₂ SO ₄ - 5.2 g / l; MgSO ₄ · 7H ₂ O - 0.55 g / l; To ₂ НРО ₄ - 1.5 g / l, water - up to 2 l pH of the nutrient medium 4.0. The cultivation temperature is 30 ° C. The fermentation process is carried out with control of pH, temperature and the rate of evolution of CO ₂ (fermentation activity). After the cessation of CO ₂ emission (after 72 hours), which indicates the end of the fermentation process, the volumetric alcohol content in the mash and sugar conversion are determined.

Post-fermentation liquid is separated from the biocatalyst granules and sent to distillation and distillation, and a nutrient medium is again fed into the reactor with the remaining catalyst and the digestion process is carried out.

The distillation and rectification of post-fermentation liquid is carried out by any known method with the production of ethyl alcohol of the required density.

The table below shows the main characteristics of the fermentation process of example 2.

Type of biocatalyst Maximum fermentation activity, ml СО ₂ / (l of medium · hour) Sugar Conversion,% Ethanol yield,% of theoretical The final alcohol content,% IGD 956 95.4 92.3 7.3

Thus, the method according to the invention allows to increase the activity and service life of the biocatalyst due to the absence of an agglomeration process while simplifying the process technology by reducing its stages.

Claims (1)

A method of producing a biocatalyst of alcoholic fermentation, including the growth of yeast biomass and its immobilization by incorporation into a gel matrix by mixing with a solution of a gel-forming material followed by its curing with Ca ⁺⁺ ions, characterized in that a mixture of exopolysaccharide with a molecular weight of 1.5 · is used as a gel-forming material 10 ⁵ -2.5 · 10 ⁵ Da obtained from the bacterial strain Paracoccus denitrificans VKPM B-8617, and sodium alginate in a mass ratio of 0.1-0.9: 3.5-4.5.