In the manufacture of any modern wine, wine yeast is necessarily used. They go through the following stages in their development:

1. Lag stage. It begins from the moment when the yeast grains enter the wort - into the nutrient medium. Cells begin to adapt to the substrate. They increase in size, but at the same time there is no reproduction process yet;
2. The second stage is called logarithmic. During it, the cell population increases, and the biomass becomes larger. Cells endure all negative environmental factors. Alcohol fermentation begins;
3. The third stage is called stationary. Yeast cells stop growing, and alcoholic fermentation occurs with intense force;
4. The fourth stage is the attenuation of the growth of yeast mass cells. The mass begins to decrease in size due to intensive autolysis and the use of reserve substances by yeast.

Having passed all four stages, the yeast mass will make any wine tasty and aromatic.

All about wine yeast

In nature, yeast forms on the surface of berries, such as grapes. They can be easily seen, as they have a light coating on the peel of the berries. Plaque is formed due to the work of a yeast fungus.

Baking, alcohol, beer and wine yeast grains are classified as industrial yeast. Given the place of origin, the grape variety and the location of the vineyards, each type of yeast is assigned its own name. Yeast races, in turn, can be divided into groups. As a result, the races of wine yeast are:

1. High fermentation;
2. Heat-resistant or cold-resistant;
3. Alcohol resistant;
4. Sherry.

Alcohol-resistant yeast races are used to make champagne, and sherry to give wines a unique aroma and taste.

Wine is usually made from the juice of grapes or other types of fruits and berries.

If artisanal winemaking takes place, the must (squeezed juice) begins to ferment without the help of yeast, as yeast fungi that are present on the surface of the berries themselves begin to multiply intensively. At the same time, lactic acid, acetic acid bacteria, yeast-like fungi come into force, which can lead to spoilage of the product, or to the production of wine vinegar instead of wine.

For this reason, during the industrial production of wine, in order to avoid spoilage of wine materials, an activated mixture of wine yeast is added to the grape juice.

The type of wine depends on how the fermentation takes place. Thanks to wine yeast, sugar, which is part of the grapes, begins to ferment. Fermentation continues until all the sugar has been converted.

With a lack of oxygen, due to the influence of yeast, alcohol is obtained. If oxygen is constantly supplied, sugar is completely oxidized and water with carbon dioxide is obtained.

During the initial stages of yeast development, fermentation occurs intensively, because of this, the carbon dioxide that is released does not allow atmospheric oxygen to penetrate to the surface of the wort. When fermentation is over, it is important to seal the barrel of wine well. If this is not done, the acetic acid bacteria will convert the alcohol into acetic acid. Instead of wine, you will become the owner of wine or apple cider vinegar.

In the industrial production of wines, grape juice with a sugar content of 25 percent is used.

To obtain white wines, the grapes are peeled and pitted. For red wines, the skins and pits are not removed. Yeast for wine, along with sugar during fermentation, juice is processed into alcohol. Yeast substances give the wine aroma and pleasant taste. After fermentation, lactic acid bacteria play an important role in giving the drink a smell.

Different varieties of wines have their own characteristics of production. For example, to get champagne, fermented wine must be fermented again. The fermentation of the drink must end in a closed container, as carbon dioxide must accumulate inside.

To get a strong wine (sherry), you need to use special sherry yeast, which is resistant to a high concentration of alcohol in the wine material.

Varieties of wines

Wines are dry, sweet and fortified. To get a dry wine, it is important to stop fermentation immediately after the end of the supply of sugar in the squeezed grape juice.

Sweet wines are made by partially fermenting sugar when a toxic alcohol level for wine yeast is reached.

Fortified wines are additionally filled with alcohol.

From the above, we can conclude that the type of wine directly depends on how it is produced, as well as what type of wine yeast is used to ferment the juice.

What are yeast

There are many different types of wine yeast. For example, wine yeast Lalvin KV-1118, Lalvin EC-1118 and others. Let's take a closer look at the instructions for using each type of yeast.

First view

Wine yeast Lalvin KV-1118 is a pure, highly active yeast concentrate that is used to make light white wines, red wines and champagnes. Also, with the help of such yeast, fermentation can be restored.

Yeast mass is usually used at low concentration, low temperatures, low content of fatty acids. They do an excellent job with their mission in a temperature regime of 10 - 35 degrees. If water is added to the wine material at a temperature below 16 degrees, esters will begin to be produced, which will give the drink a rich aroma. Due to the pronounced killer effect, yeast grains well suppress the "wild" microflora.

Instructions for use of such a product says the following:

1. KV-stamped yeast is used to express grape aroma in white, rosé and deep red wines;
2. Given the type and purity of raw materials, the conditions and duration of fermentation, the required dosage is determined. Usually it is from 1 to 4 g/dal;
3. They do not contain any additives. They have a moisture content of 6 percent;
4. Wine yeast (5 grams) is diluted in water (50 milliliters) 34 - 39 degrees. In order for them to work properly, it is important that the water temperature is no more than 40 degrees. Then the mixture must be mixed well to break up the lumps and withstand no more than twenty minutes. After a while, mix again, and slowly pour into the wort. Slow introduction helps the yeast to gradually acclimatize and not die when combined with cool wort;
5. Wine yeast can be stored in a dark, dry place for up to a couple of years. Storage temperature should be between five and fifteen degrees. If you open the package, it has a shelf life of no more than six months.

Second view

Wine yeast mass Lalvin EC gives red and white wines a refreshing taste and purity. They ferment well even at the lowest temperatures, forming a sediment in one place. Thanks to this type of raw material, fermentation can be restarted. It is recommended to use it for, as well as from viburnum, hawthorn and cherries. An EC-labeled product has low foaming, clarifies wine well and collects sediment compactly. The instructions for use of EC stamped yeast say the following:

1. 300 grams of the contents of the bag should be poured into five liters of forty-degree water. Stir thoroughly until smooth;
2. When the temperature of the mixture reaches 35 degrees, carefully pour 250 grams of yeast onto the surface. Let stand 20 minutes and mix well. Then pour the resulting mass into the wort, so that the temperature difference is not higher than ten degrees;
3. You can store them in a closed package at a temperature of no more than eight degrees Celsius.

Making wine from grapes is not very difficult. It is only important to purchase the right yeast and carefully study what the instructions say. It usually has everything written on it.

Now you know what wine yeast is. What types are they. How can you get different types of wines using different types of production. Wine lovers are always proud of their creations, especially if people around them like them.

... of the fermentation stand out on the surface of the fermented medium in the form of a rather thick layer of foam and remain in this state until the end of fermentation. They then settle, but rarely form a dense sediment at the bottom of the fermentation vessel. Top-fermenting yeast is structurally a pulverized yeast that does not stick together, unlike bottom-fermenting flaky yeast, whose shells are sticky, which leads to agglutination and rapid cell settling.

Bottom-fermenting yeast, developing in the fermented liquid, does not pass into the surface layer - foam, quickly settles at the end of fermentation, forming a dense layer at the bottom of the fermentation vessel.

A distinguishing feature is the ability of bottom-fermenting yeasts to completely ferment raffinose, while most top-fermenting yeasts do not break down raffinose at all, and only a few species can only ferment it by one third. This main difference is explained by the fact that α-galactosidase is contained in the enzyme complex of this type of yeast.

Of the cultural yeasts, bottom-fermenting yeasts include most wine and beer yeasts, and top-fermenting yeasts include alcohol, baker's, and some races of brewer's yeast. Initially, only top-fermenting yeast was known, since the fermentation of all juices occurred at ordinary temperatures. Wanting to get drinks saturated with CO 2, a person began to ferment at a low temperature. Under the influence of changed external conditions, bottom-fermenting yeast with its properties has become widespread.

In addition to general properties, the yeast used in a particular production has specific characteristics. Moreover, in the same production, varieties are used that differ in one or more features. They are taken out of one cage. Such cultures are called races (strains). Each production has several races of yeast.

Races of alcohol production yeast

In alcohol production, those top-fermenting yeast races are used that have the highest fermentation energy, form a maximum of alcohol and ferment mono- and disaccharides, as well as part of dextrins. Of the yeast used in the production of alcohol from bread and potato raw materials, races HP, M and XV should be mentioned.

When processing molasses into alcohol, races I, L, V, G-67, G-73 are used. These races belong to the family Saccharomyces taceae, genus Saccharomyces, species cerevisiae.

The HP race was isolated in 1902 from compressed baker's yeast. Yeast cells of this race are round, ovoid, 5-6.2 x 5-8 microns in size.

The development and reproduction of HP yeast race is very fast. They ferment glucose, fructose, sucrose, galactose, maltose, mannose, raffinose by one third and can form up to 13% alcohol in the fermented medium.

Race M (Mischung - mixture), proposed by Genneberg in 1905, consists of a mixture of four races of top-fermenting yeast; it is intended for the fermentation of media containing a mixture of various sugars (dextrins, raffinose), which are fermented differently by different yeasts. Such a mixed culture is very resistant to various abnormal conditions encountered in factory practice.

Race XV is technologically similar to race HP. It is used along with the HP race for the fermentation of mixed grain-molasses raw materials.

Of these races, the HP race, which is also used in hydrolysis and sulfite-alcohol production, is the most suitable for fermenting wort from starchy raw materials. However, for the fermentation of sulfite liquors, sulfite yeasts have been specially bred to ferment glucose, fructose, galactose and mannose.

Yeast used in distilleries processing molasses must have a specific ability to quickly ferment quite concentrated sugar solutions and to tolerate high salt content in the medium well. So-called osmophilic yeasts, which tolerate very high osmotic pressure, can ferment solutions containing high concentrations of sugar.

Race Y, bred from molasses yeast by K.Yu. Yakubovsky. Race I has an exceptional ability to ferment high concentrations of sugar and tolerates high levels of salts and alcohol in the fermented molasses wort. Yeast race I ferment glucose, fructose, sucrose, galactose, maltose; raffinose is fermented only partially and dextrins and lactose are not fermented at all. Race I refers to top-fermenting pulverized yeast.

The yeast of race L (Lokhvitskaya) is close in its properties to the yeast of race I, but they reproduce somewhat better and ferment sugar more completely.

Race B (Hungarian), like race L, is adapted to a molasses environment. These races ferment well sucrose, glucose, fructose, and partially raffinose.

Yeast races L and B, along with high fermentation properties, also have good lifting power (the ability to raise the dough), which allows them to be separated from the mash and produced in a pressed form as a bakery.

Successful use is being made of hybrid yeasts bred at the Institute of Genetics of the USSR Academy of Sciences by crossing two types of yeast. Among the hybrids, G-67, G-73 are of the greatest interest. Hybrid 67 was obtained by crossing brewer's yeast S-carlsbergensis with S. cerevisiae of race I. Further crossing of hybrid 67 with hybrid 26 (obtained from crossing races I and HP) gave hybrid 73. Hybrids 67 and 73, along with other enzymes, contain α-galactosidase and possess ability to complete fermentation of raffinose. Other hybrid yeasts are also recommended for use.

Races of baker's yeast

In the yeast industry, fast-growing yeast races with good buoyancy and good storage stability are valued. The taste of baker's yeast should be pure, white or yellowish in color. The lifting force is determined both by the characteristics of the yeast races and by the method of production. Yeast resistance is a property of the race, but depends on the internal state of the cells and the purity of the yeast.

In the production of baker's yeast from molasses, races VII, 14, 28 and G-176 are used.

Race VII, bred from pressed commercial yeast from the Tomsk Yeast Plant, multiplies rapidly and is well pressed to a moisture content of 71-72%. Race VII yeasts have good buoyancy and the highest storage stability compared to other yeasts known in factory practice. In addition, this culture is resistant to harmful impurities contained in molasses.

Race 14 is intended for the production of dry yeast. This yeast is characterized by a dense texture at a moisture content of 75%, high thermal stability.

From the hybrids of baker's yeast, hybrid 176 was selected, which has all the positive features: large cells (5.6-14.0 microns), resistance to harmful impurities of molasses and a high multiplication factor, which is higher in this race than in the most rapidly breeding race 14. Other promising hybrid races of yeast are currently undergoing production tests.

Races of brewer's yeast

In brewing, bottom-fermenting yeast is used, adapted to relatively low temperatures. Brewer's yeast must be microbiologically pure, as well as have the ability to flocculate, quickly settle to the bottom of the fermenter and give a clear drink with a certain taste and aroma. Highly fermenting and easy flaking yeasts include Froberg bottom-fermenting brewer's yeast (Saccharomyces cerevisiae Froberg), yeast races V and 776.

In breweries, yeast of the 776 race, which was bred at the beginning of the 20th century, was widely used. This yeast is considered particularly suitable for the fermentation of wort prepared with the addition of unmalted materials or from malted barley with a low degree of germination. Yeast of race 776 is medium-fermenting, during the period of main fermentation on wort with a concentration of 11%, they form approximately 2.7% CO 2 . Cells are ovoid, 8-10 µm long and 5-6 µm wide. Yeast mass gain 1: 5.4. The lightening ability is satisfactory.

Of the other yeasts, breweries use races 11, 41, 44, S-Lvovskaya and others, which differ in fermentation energy, sedimentation ability and growth energy.

Race 11 yeast is highly fermentative with good clarification capability. Beer made with race 11 yeast tastes good. This race has become widespread in breweries.

Yeast of race 41 is medium fermenting, with good sedimentation ability. When the wort is fermented with race 41, a mild beer with a clean taste is obtained.

Race 44 yeast is medium fermenting. Sedimentation ability is good. They give beer fullness of taste and give good results when used in the production of water with increased hardness.

Race S yeast is medium fermenting. Sedimentation ability is good. Give beer with a mild clean taste.

Race P yeast is medium-fermenting, well clarifies beer and determines a pleasant clean taste.

Race F yeast is characterized by a good clarification ability and imparts a pleasant aroma to the beer. The race is resistant to the action of foreign microorganisms.

Yeast of race A (isolated at the Riga brewery "Aldaris") ferments the wort in 7-8 days, clarifies beer well and is resistant to infection.

A number of strongly fermenting yeast strains (28, 48, 102) were obtained by different selection methods at the All-Russian Research Institute of the Beer and Non-Alcoholic Industry, which have a significantly higher fermentation energy than the yeast of the original race 11.

Top-fermented brewer's yeast is widely used in England in the preparation of Porter. They are also used to make Berlin lager beers and other drinks. For the preparation of Velvet beer, strain 191 K is used, which intensively ferments monosaccharides and maltose, but does not ferment sucrose, raffinose and lactose.

Wine yeast races

In winemaking, yeasts are valued for their rapid multiplication, the ability to suppress other types of yeast and microorganisms and give the wine an appropriate bouquet. The yeast used in winemaking belongs to a peculiar species of Saccharomyces ellipsoideus. Their cells are oblong-oval in shape. Yeast vigorously ferments glucose, fructose, sucrose and maltose. In different localities and from different young wines, several distinct varieties or races of this species have been isolated. In winemaking, almost all yeast production cultures are of their own, local origin. These include races Magarach 7, Massandra 3, Pino 14, Kakhuri and many others. Along with these races, some foreign ones are also used, for example, the Steinberg race, isolated in Germany in 1892 and 1893, and the Champagne-Ai race.

Most wine yeast is a bottom-fermenting yeast.

For the preparation of white table wines, the races Pinot 14, Feodosia 1/19, Aligote, Riesling Anapsky are used.

Race Pinot 14 has egg-shaped cells, well ferments grape must with a sugar content of 20% with the formation of 11.57% alcohol by volume; the optimum temperature for development and fermentation is 18: -25°C. This race is cold and acid resistant; the optimal pH value is 2.9-3.9.

Race Theodosius 1/19 - large-celled, pulverized, very energetic, quickly ferments grape must and ferments it well; has a wide fermentation temperature range (from 9 to 35°C) and can be used as cold-resistant and heat-resistant.

There are several races of Aligote yeast, and they are all strong, with high fermentation energy. Riesling Anapsky yeast also belongs to vigorous fermenters.

For the preparation of strong wines, the Massandra 3 race is used with an ovoid cell shape, pulverized; the optimal pH value is 3.7-4.05; the optimum fermentation temperature is 18-20°C. Grape must with a sugar content of 20% is completely fermented; when fermenting concentrated grape must (30% sugar), it forms 11.8% alcohol by volume and leaves 8.7% sugar unfermented.

Race Magarach 125, named to commemorate the 125th anniversary of the first planting of grapes at the Magarach Institute, is used to produce strong and dessert wines. This race well ferments highly concentrated grape must with a sugar content of 27-30%, cold-resistant.

Race Kakhuri 2 is widely used for the preparation of champagne wine materials and wines. It ferments grape must with a sugar content of 20% with the formation of 11.4% alcohol, 0.28% sugar remains unfermented. This race is quite cold-resistant (at a temperature of 14-15 ° C, the must ferments on the 2nd day) and ferments well; the optimal pH value is 3.4-3.6.

Race Champagne 7, used for champagnization of bottled wine, is isolated from race Kakhuri 5 and is characterized by the formation of a sediment that is difficult to stir up; intensively ferments at a temperature of 4-9°C, although the wort ferments only on the 5th-6th day.

Of the wine yeasts, the Leningradskaya race is considered the most cold-resistant, and the Ashgabat 3 race is considered the most heat-resistant.

In the production of sherry, special races of yeast are used, which are a variety of the Saccharomyces oviformis species. Sherry yeast forms a film on the surface of wine in incomplete barrels, thanks to the development of which the wine acquires a special bouquet and taste.

Through careful selection of the most important production characteristics, several races of sherry yeasts (13, 15 and 20) with high film-forming ability have been identified. Later, from the production that used the Sherry 20 race, a more effective Sherry 20-C race was selected, which was widely used in many sherry factories.

In fruit and berry winemaking, selected races of yeast isolated from various fruit and berry juices are used. Fruit and berry juices are rich in yeast, which has all the qualities necessary for production and is biologically adapted to the conditions of development in the original fruit and berry juices. Therefore, yeast strains isolated from strawberry juices are used to ferment strawberry juices, and yeast strains isolated from cherry juices are used to ferment cherry juices, etc.

The following strains have become widespread in fruit and berry winemaking: apple 46, 58, cranberry 17, currant 16, lingonberry 3, 7, 10, raspberry 7/5, 25, 28, 28/10, cherry 3, 6, strawberry 7, 4 , 9.

These yeast strains ensure the normal course of fermentation, the completeness of fermentation, rapid clarification and good taste of the wine; they ferment glucose, fructose, sucrose, maltose, galactose and do not ferment lactose and mannitol.

Yeast races Moscow 30, Apple 7, Cherry 33, Chernomorodinovaya 7, Raspberry 10 and Plum 21 are successfully used in fruit and berry winemaking. Pure yeast culture Moscow 30 is recommended for fermentation of cranberry must; Apple 7 and Cherry 33 - for the fermentation of apple must; Blackcurrant 7 and Cherry 33 - for the fermentation of blackcurrant and cherry must.

4 Chemistry of alcoholic fermentation. Secondary and by-products of alcoholic fermentation

Alcoholic fermentation is a chain of enzymatic processes, the end result of which is the breakdown of hexose with the formation of alcohol and CO 2 and the delivery to the yeast cell of the energy that is necessary for the formation of new substances used for life processes, including growth and reproduction. By chemical nature, alcoholic fermentation is a catalytic process that occurs under the action of biological catalysts - enzymes.

The modern theory of alcoholic fermentation is the result of the work of many scientists from around the world.

For elucidation of the processes of fermentation, the works of outstanding Russian scientists were of great importance: Lebedev, Kostychev, Favorsky, Ivanov, Engelhardt.

According to modern concepts, alcoholic fermentation is a complex continuous process of sugar breakdown, catalyzed by various enzymes with the formation of 12 intermediate products.

1 The initial stage of glucose conversion is the reaction of its phosphorylation with the participation of the enzyme glucosinase. A phosphate residue from the ATP molecule, which is located in yeast cells, is attached to the glucose molecule, and glucose-6-phosphate is formed, and ATP is converted to ADP:

C 6 H 12 O 6 + ATP → CH 2 O (H 2 PO 3) (CHOH) 4 CHO + ADP

Glucose Glucose-6-phosphate

As a result of the addition of a phosphate residue from the ATP molecule to glucose, the reactivity of the latter increases.

2 Glucose-6-phosphate, by isomerization under the action of the enzyme glucose phosphate isomerase, is converted reversibly into the form of fructose:

CH 2 O (H 2 RO 3) (CHOH) 4 CHO → CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 OH

Glucose-6-phosphate Fructose-6-phosphate

CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 OH + ATP →

Fructose 6-phosphate

→ CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 O (H 2 RO) + ADP

Fructose 1,6-diphosphate

Esters of glucose-6-phosphate and fructose-6-phosphate form an equilibrium mixture, called the Emden ester and consisting of 70-75% Robison ester (glucose) and 25% Neuberg ester (fructose).

The formation of fructose-1,6-diphosphate ends the preparatory the stage of alcoholic fermentation with the transfer of high-energy phosphate bonds and with the transformation of hexose into a labile oxyform, which is easily subjected to further enzymatic transformations.

4 The next most important step is desmolysis - breaking the carbon chain of fructose diphosphate with the formation of two
phosphotriosis molecules. The symmetrical arrangement of phosphoric acid residues at the ends of the fructose molecule makes it easier to break its carbon chain right in the middle. Fructose diphosphate decomposes into two trioses: phosphoglyceraldehyde and phosphodioxyacetone. The reaction is catalyzed by the enzyme aldolase and is reversible:

CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 O (H 2 RO) → CH 2 O (H 2 P0 3) COCH 2 OH +

Fructose 1,6-diphosphate Phosphodioxyacetone

CH 2 0 (H 2 ROz) SUPERB (4)

3-phosphoglyceraldehyde

The main role in further transformations during alcoholic fermentation belongs to 3-phosphoglyceraldehyde, but in the fermented liquid it is found only in small quantities. This is due to the mutual transition of the ketose to aldose isomer and back under the action of the enzyme triose phosphate isomerase (5.3.1.1)

CH 2 0 (H 2 P0 3) COCH 2 OH; £ CH 2 0 (H 2 P0 3) SWEET

Phosphodioxyacetone 3-Phosphoglyceraldehyde

As phosphoglyceraldehyde is further converted, new amounts of it are formed during the isomerization of phosphodioxyacetone.

5. The next step is the oxidation of two molecules of 3^phosphoglyceraldehyde. This reaction is catalyzed by triose phosphate dehydrogenase (1.2.1.12), whose coenzyme is NAD (nicotinamide adenine dinucleotide). The phosphoric acid of the medium is involved in the oxidation. The reaction proceeds according to the following equation: 2CH 2 0 (H 2 P0 3) SHORTLY + 2H 3 P0 4 + 2NAD Triose phosphate dehydrogenase ->

3-phosphoglyceraldehyde

->- 2CH 2 0 (H 2 P0 3) SNONSOO w (H 2 P0 3) + 2NAD

1,3-diphosphoglyceric acid

The 3-phosphoglyceraldehyde molecule adds phosphate, and the hydrogen is transferred to the NAD coenzyme, which is reduced. The energy released as a result of the oxidation of 3-phosphoglyceraldehyde is accumulated in the macroergic bond of the resulting 1,3-diphosphoglycerol

1,3-diphosphoglyceric acid 3-phosphoglyceric acid

7. Then, under the action of the enzyme phosphoglyceromutase
(2.7.5.3) the phosphoric acid residue moves from the third
carbon to the second, and as a result 3-phosphoglyceric acid
lota is converted to 2-phosphoglyceric acid:

2CH 2 (H 2 P0 3) CHOHCOOH ^t 2CH 2 0HCH0 (H 2 P0 3) COOH. (7)

3-phosphoglyceric acid 2-phosphoglyceric acid

8. The next step is the dephosphorylation of 2-phospho-
foglyceric acid. At the same time, 2-phosphoglycerol acid
by the action of the enzyme enolase (4.2.1.11) by dehydro
tiation (loss of water) is converted into phosphoenolpyrovino-
gradic acid:

2CH 2 OHCHO (H 2 P0 3) COOH qt 2CH 3: CO co (H 2 P0 3) COOH + 2H 2 0. (8)

2-phosphoglyceric acid Sosphoenolpyruvic acid

During this transformation, the intramolecular energy is redistributed and most of it is accumulated in the macroergic phosphate bond.

9. Very unstable phosphoenolpyruvic acid
easily dephosphorylated, while the phosphoric acid residue
by the action of the enzyme pyruvate kinase (2.7.1.40)
together with a macroergic bond to the ADP molecule. As a result
a more stable keto form of pyruvic acid is formed
you, and ADP is converted to ATP:

2CH 2: CO syu (H 2 P0 3) COOH + 2ADP - * 2CH 3 COCOOH + 2ATP. (3)

Phosphoenol pyruvic pyruvic

acid acid

10. Pyruvic acid under the action of the enzyme pi-
ruvate decarboxylase (4.1.1.1) is decarboxylated to cleave
nii CO 2 and the formation of acetaldehyde:

2CH 3 COCOOH - * 2C0 2 + 2CH 3 CHO. (ten)

pyruvic aldehyde

11. Acetic aldehyde with the participation of the enzyme alcohol dehy-
rogenase (1.1.1.1) interacts with NAD-H 2 formed
earlier, during the oxidation of phosphoglyceraldehyde to phospho-
glyceric acid [see equation (5)]. As a result, vinegar
aldehyde is reduced to ethyl alcohol, and the coenzyme
NAD-H 2 is regenerated again (oxidized to NAD):

2SN 3 CHO + 2NAD H 2 Z 2CH 3 CH 2 OH + 2OVER. (eleven)

So, the final stage of fermentation is the reduction reaction of acetaldehyde to ethyl alcohol.

From the considered cycle of alcoholic fermentation reactions, it can be seen that 2 alcohol molecules and 2 CO 2 molecules are formed from each glucose molecule.

In the process of alcoholic fermentation, four ATP molecules are formed [see. equations (6) and (9)], but two of them are spent on phosphorylation of hexoses [see. equations (1) and (3)]. Thus, only 2 g-mol of ATP is stored.

It was previously indicated that 41.9 kJ is spent on the formation of each gram-molecule of ATP from ADP, and 83.8 kJ, respectively, goes into the energy of two ATP molecules. Therefore, during the fermentation of 1 g-mol of glucose, the yeast receives an energy of about 84 kJ. This is the biological meaning of fermentation. With the complete breakdown of glucose into CO 2 and water, 2874 kJ is released, and when 1 g-mol of glucose is oxidized to CO 2 and H 2 0, 2508 kJ is accumulated during aerobic respiration, since the resulting ethanol still retains potential energy. Thus, from an energy point of view, fermentation is an uneconomical process.

The fermentation of individual sugars occurs in a certain sequence, determined by the rate of their diffusion into the yeast cell. Glucose and fructose are the fastest fermented by yeast. However, sucrose as such disappears into the must (is inverted) at the beginning of fermentation. It is hydrolyzed by p-fructofuranosidase (3.2.1.26) of the yeast cell membrane to form hexoses (glucose and fructose), which are easily used by the cell. When there is almost no fructose and glucose left in the wort, the yeast begins to consume maltose.

§ 5. SECONDARY AND BY-PRODUCTS OF ALCOHOLIC FERMENTATION

All substances resulting from the fermentation of sugar by yeast, with the exception of alcohol and CO 2, are secondary products of alcoholic fermentation. In addition to them, there are by-products of alcoholic fermentation, which are formed not from sugar, but from other substances in the fermented substrate. These include amyl, isoamyl, iso-butyl and other alcohols known as fusel oil.

Of the secondary products of alcoholic fermentation, glycerin, acetaldehyde, pyruvic, acetic, succinic, citric and lactic acids, acetoin (acetylmethyl-carbinol), 2,3-butylene glycol and diacetyl are known. Under aerobic conditions, pyruvic acid is also the starting material for the tricarboxylic acid cycle (Krebs cycle), according to which acetic, citric, malic, and succinic acids are formed from it. Higher alcohols are also formed from pyruvic acid by amination to alanine, which in turn is transaminated into the corresponding keto acid. Under conditions of alcoholic fermentation, keto acids, being reduced, form higher alcohols. Therefore, secondary and by-products of alcoholic fermentation cannot be strictly distinguished.

Acetic aldehyde can undergo dismutation with the formation of acetic acid and ethyl alcohol (Cannizzaro reaction):

CH 3 CH + CH 3 CH + H 2 0 \u003d CH3COOH + CH 3 CH 2 OH.

One of the aldehyde molecules is oxidized to an acid, while the other is reduced to an alcohol. In an alkaline environment, one molecule

acetaldehyde enters into a redox reaction with the second molecule of acetaldehyde; in this case, ethyl alcohol, acetic acid and, simultaneously with them, glycerin are formed, which is expressed by the following total equation:

2C 6 Hi 2 0 6 + H 2 0 \u003d 2CH 2 OHCHNOCH 2 OH + CH 3 CH 2 OH + CH 3 COOH + 2C0 2.

Glycerin is formed in a small amount during alcoholic fermentation. If the fermentation conditions change, its production can be carried out on an industrial scale.

Glycerin and acetaldehyde are intermediate products of alcoholic fermentation. At the last stage of the normal fermentation process, a significant part of the acetaldehyde is reduced to ethanol. But if acetaldehyde is bound with sodium sulfite, then the direction of alcoholic fermentation will change towards the formation of large amounts of glycerin.

The removal of acetaldehyde from the fermented medium with sodium sulfite is presented as follows:

CH 3 CHO + Na 2 S0 3 + H 2 OW CH 3 CHONaHS0 2 + NaOH.

Acetic aldehyde, formed during the decarboxylation of pyruvic acid, cannot serve as a hydrogen acceptor as a result of binding with sulfite. The place of acetic aldehyde is occupied by phosphodioxyacetone, which receives hydrogen from the reduced NAD-H 2, forming a-glycerophosphate. This reaction is catalyzed by the enzyme glycerophosphate dehydrogenase. Under the action of phosphatase, α-glycerophosphate is de-phosphorylated, turning into glycerol. Thus, in the presence of Na 2 S03, glycerol-aldehyde fermentation proceeds:

C 6 H 12 0 6 \u003d CH3CHO + CH 2 OHCHNOCH 2 OH + C0 2.

Sugar Acetaldehyde Glycerin

With an increase in the amount of sodium sulfite introduced into the fermented medium, the amount of bound aldehyde increases accordingly and the formation of ethanol and CO 2 is weakened.

Formation of acids and acetoin. Succinic acid is formed by dehydrogenation and condensation of two molecules of acetic acid with one molecule of acetaldehyde (hypothesis V. 3. Gvaladze and Genavua):

2CH 3 C00H + CH 3 CHO -* C00HCH 2 CH 2 C00H + CH 3 CH 2 OH.

In the process of alcoholic fermentation, succinic acid is also formed by deamination of glutamic acid. The hydrogen acceptor in this reaction is trioseglycerol aldehyde, so the deamination reaction is accompanied by the simultaneous accumulation of glycerol:

C 6 Hi 2 0 6 + COOHCH2CH2CHNH2COOH + 2H 2 0 \u003d CO0HCH 2 CH 2 COOH -b

Glucose Glutamic acid Succinic acid

2CH 2 OHCHNOCH 2 OH 3 + NH 3 + CO 2.

Glycerol

Ammonia is consumed by yeast for protein synthesis, while glycerol and succinic acid are released into the medium.

The formation of citric acid, according to Lafon, comes from. nine molecules of acetaldehyde:

9CH 3 COOH + 4H 2 0 \u003d (CH 2 COOH) 2 C (OH) COOH + 6CH 3 CH 2 OH.

Lemon acid

The formation of lactic acid is explained by the reduction of pyruvic acid:

CH3SOCOOH + H 2 -> CH 3 CH (OH) COOH.

Pyruvic Lactic Acid

However, its formation is considered more likely as a result of the hydrolysis of an intermediate product of alcoholic fermentation - phosphoglyceraldehyde:

SNOSNONSN 2 OP0 3 H 2 + H 2 0 - * CH 3 CH (OH) COOH + H 3 P0 4.

Phosphoglycerol Lactic Acid

aldehyde

The condensation of acetic acid with acetaldehyde explains the formation of acetoin:

1) CH3COOH + CH 3 CHO->-CH3COCOCH3 + H 2 0;

Diacetyl

2) CH3COCOCH3 + CH3CHO -4 CH3COCHOHCH3 + CH3COOH.

First, diacetyl is formed; then, by dismutation of the conjugated redox with diacetyl water, acetoin is formed.

When acetoin is reduced, 2,3-butylene glycol is formed:

CH 3 SOSNONSNz + OVER ■ H 2 *± CH 3 CHONSNONCH 3 + OVER.

The mechanism of formation of some secondary products of alcoholic fermentation is not yet entirely clear, but there is no doubt that acetaldehyde is the main starting material for the synthesis of secondary fermentation products.

Among the secondary products, acetic and succinic acids predominate, as well as 2,3-butylene glycol and acetic acids.

2 General characteristics and races of yeast used in fermentation industries
Cultural yeast belongs to the Saccharomycetes family and is called Saccharomyces cerevisiae.

The optimum temperature for yeast propagation is in the range of 25-30°C, and the minimum temperature is about 2-3°C. At a temperature of 40 ° C, growth stops and the yeast dies, but the yeast tolerates low temperatures well, although their reproduction stops. Yeast does not die even at -180°C (liquid air). At a high concentration of sugar in the medium, the vital activity of the yeast stops, as this increases the osmotic pressure, at a certain value of which plasmolysis of the yeast cells occurs. Plasmolysis is called contraction of the cell, followed by exfoliation of the protoplasm from the cell membrane due to dehydration of the cell and the associated sharp drop in cell sap pressure. The value of the maximum concentration of sugar for different races of yeast is not the same.

There are top and bottom fermenting yeasts. Within each of these groups there are several distinct races.

Top-fermenting yeast in the stage of intensive fermentation stand out on the surface of the fermented medium in the form of a rather thick layer of foam and remain in this state until the end of fermentation. They then settle, but rarely form a dense sediment at the bottom of the fermentation vessel. Top-fermenting yeast is structurally a pulverized yeast that does not stick together, unlike bottom-fermenting flaky yeast, whose shells are sticky, which leads to agglutination and rapid cell settling.

Bottom-fermenting yeast, developing in the fermented liquid, does not pass into the surface layer - foam, quickly settles at the end of fermentation, forming a dense layer at the bottom of the fermentation vessel.

A distinguishing feature is the ability of bottom-fermenting yeasts to completely ferment raffinose, while most top-fermenting yeasts do not break down raffinose at all, and only a few species can only ferment it by one third. This main difference is explained by the fact that α-galactosidase is contained in the enzyme complex of this type of yeast.

Of the cultural yeasts, bottom-fermenting yeasts include most wine and beer yeasts, and top-fermenting yeasts include alcohol, baker's, and some races of brewer's yeast. Initially, only top-fermenting yeast was known, since the fermentation of all juices occurred at ordinary temperatures. Wanting to get drinks saturated with CO 2, a person began to ferment at a low temperature. Under the influence of changed external conditions, bottom-fermenting yeast with its properties has become widespread.

In addition to general properties, the yeast used in a particular production has specific characteristics. Moreover, in the same production, varieties are used that differ in one or more features. They are taken out of one cage. Such cultures are called races (strains). Each production has several races of yeast.
Races of alcohol production yeast

In alcohol production, those top-fermenting yeast races are used that have the highest fermentation energy, form a maximum of alcohol and ferment mono- and disaccharides, as well as part of dextrins. Of the yeast used in the production of alcohol from bread and potato raw materials, races HP, M and XV should be mentioned.

When processing molasses into alcohol, races I, L, V, G-67, G-73 are used. These races belong to the family Saccharomyces taceae, genus Saccharomyces, species cerevisiae.

The HP race was isolated in 1902 from compressed baker's yeast. Yeast cells of this race are round, ovoid, 5-6.2 x 5-8 microns in size.

The development and reproduction of HP yeast race is very fast. They ferment glucose, fructose, sucrose, galactose, maltose, mannose, raffinose by one third and can form up to 13% alcohol in the fermented medium.

Race M (Mischung - mixture), proposed by Genneberg in 1905, consists of a mixture of four races of top-fermenting yeast; it is intended for the fermentation of media containing a mixture of various sugars (dextrins, raffinose), which are fermented differently by different yeasts. Such a mixed culture is very resistant to various abnormal conditions encountered in factory practice.

Race XV is technologically similar to race HP. It is used along with the HP race for the fermentation of mixed grain-molasses raw materials.

Of these races, the HP race, which is also used in hydrolysis and sulfite-alcohol production, is the most suitable for fermenting wort from starchy raw materials. However, for the fermentation of sulfite liquors, sulfite yeasts have been specially bred to ferment glucose, fructose, galactose and mannose.

Yeast used in distilleries processing molasses must have a specific ability to quickly ferment quite concentrated sugar solutions and to tolerate high salt content in the medium well. So-called osmophilic yeasts, which tolerate very high osmotic pressure, can ferment solutions containing high concentrations of sugar.

Race Y, bred from molasses yeast by K.Yu. Yakubovsky. Race I has an exceptional ability to ferment high concentrations of sugar and tolerates high levels of salts and alcohol in the fermented molasses wort. Yeast race I ferment glucose, fructose, sucrose, galactose, maltose; raffinose is fermented only partially and dextrins and lactose are not fermented at all. Race I refers to top-fermenting pulverized yeast.

The yeast of race L (Lokhvitskaya) is close in its properties to the yeast of race I, but they reproduce somewhat better and ferment sugar more completely.

Race B (Hungarian), like race L, is adapted to a molasses environment. These races ferment well sucrose, glucose, fructose, and partially raffinose.

Yeast races L and B, along with high fermentation properties, also have good lifting power (the ability to raise the dough), which allows them to be separated from the mash and produced in a pressed form as a bakery.

Successful use is being made of hybrid yeasts bred at the Institute of Genetics of the USSR Academy of Sciences by crossing two types of yeast. Among the hybrids, G-67, G-73 are of the greatest interest. Hybrid 67 was obtained by crossing brewer's yeast S-carlsbergensis with S. cerevisiae of race I. Further crossing of hybrid 67 with hybrid 26 (obtained from crossing races I and HP) gave hybrid 73. Hybrids 67 and 73, along with other enzymes, contain α-galactosidase and possess ability to complete fermentation of raffinose. Other hybrid yeasts are also recommended for use.
Races of baker's yeast

In the yeast industry, fast-growing yeast races with good buoyancy and good storage stability are valued. The taste of baker's yeast should be pure, white or yellowish in color. The lifting force is determined both by the characteristics of the yeast races and by the method of production. Yeast resistance is a property of the race, but depends on the internal state of the cells and the purity of the yeast.

In the production of baker's yeast from molasses, races VII, 14, 28 and G-176 are used.

Race VII, bred from pressed commercial yeast from the Tomsk Yeast Plant, multiplies rapidly and is well pressed to a moisture content of 71-72%. Race VII yeasts have good buoyancy and the highest storage stability compared to other yeasts known in factory practice. In addition, this culture is resistant to harmful impurities contained in molasses.

Race 14 is intended for the production of dry yeast. This yeast is characterized by a dense texture at a moisture content of 75%, high thermal stability.

From the hybrids of baker's yeast, hybrid 176 was selected, which has all the positive features: large cells (5.6-14.0 microns), resistance to harmful impurities of molasses and a high multiplication factor, which is higher in this race than in the most rapidly breeding race 14. Other promising hybrid races of yeast are currently undergoing production tests.

Races of brewer's yeast

In brewing, bottom-fermenting yeast is used, adapted to relatively low temperatures. Brewer's yeast must be microbiologically pure, as well as have the ability to flocculate, quickly settle to the bottom of the fermenter and give a clear drink with a certain taste and aroma. Highly fermenting and easy flaking yeasts include Froberg bottom-fermenting brewer's yeast (Saccharomyces cerevisiae Froberg), yeast races V and 776.

In breweries, yeast of the 776 race, which was bred at the beginning of the 20th century, was widely used. This yeast is considered particularly suitable for the fermentation of wort prepared with the addition of unmalted materials or from malted barley with a low degree of germination. Yeast of race 776 is medium-fermenting, during the period of main fermentation on wort with a concentration of 11%, they form approximately 2.7% CO 2 . Cells are ovoid, 8-10 µm long and 5-6 µm wide. Yeast mass gain 1: 5.4. The lightening ability is satisfactory.

Of the other yeasts, breweries use races 11, 41, 44, S-Lvovskaya and others, which differ in fermentation energy, sedimentation ability and growth energy.

Race 11 yeast is highly fermentative with good clarification capability. Beer made with race 11 yeast tastes good. This race has become widespread in breweries.

Yeast of race 41 is medium fermenting, with good sedimentation ability. When the wort is fermented with race 41, a mild beer with a clean taste is obtained.

Race 44 yeast is medium fermenting. Sedimentation ability is good. They give beer fullness of taste and give good results when used in the production of water with increased hardness.

Race S yeast is medium fermenting. Sedimentation ability is good. Give beer with a mild clean taste.

Race P yeast is medium-fermenting, well clarifies beer and determines a pleasant clean taste.

Race F yeast is characterized by a good clarification ability and imparts a pleasant aroma to the beer. The race is resistant to the action of foreign microorganisms.

Yeast of race A (isolated at the Riga brewery "Aldaris") ferments the wort in 7-8 days, clarifies beer well and is resistant to infection.

A number of strongly fermenting yeast strains (28, 48, 102) were obtained by different selection methods at the All-Russian Research Institute of the Beer and Non-Alcoholic Industry, which have a significantly higher fermentation energy than the yeast of the original race 11.

Top-fermented brewer's yeast is widely used in England in the preparation of Porter. They are also used to make Berlin lager beers and other drinks. For the preparation of Velvet beer, strain 191 K is used, which intensively ferments monosaccharides and maltose, but does not ferment sucrose, raffinose and lactose.

Wine yeast races

In winemaking, yeasts are valued for their rapid multiplication, the ability to suppress other types of yeast and microorganisms and give the wine an appropriate bouquet. The yeast used in winemaking belongs to a peculiar species of Saccharomyces ellipsoideus. Their cells are oblong-oval in shape. Yeast vigorously ferments glucose, fructose, sucrose and maltose. In different localities and from different young wines, several distinct varieties or races of this species have been isolated. In winemaking, almost all yeast production cultures are of their own, local origin. These include races Magarach 7, Massandra 3, Pino 14, Kakhuri and many others. Along with these races, some foreign ones are also used, for example, the Steinberg race, isolated in Germany in 1892 and 1893, and the Champagne-Ai race.

Most wine yeast is a bottom-fermenting yeast.

For the preparation of white table wines, the races Pinot 14, Feodosia 1/19, Aligote, Riesling Anapsky are used.

Race Pinot 14 has egg-shaped cells, well ferments grape must with a sugar content of 20% with the formation of 11.57% alcohol by volume; the optimum temperature for development and fermentation is 18: -25°C. This race is cold and acid resistant; the optimal pH value is 2.9-3.9.

Race Theodosius 1/19 - large-celled, pulverized, very energetic, quickly ferments grape must and ferments it well; has a wide fermentation temperature range (from 9 to 35°C) and can be used as cold-resistant and heat-resistant.

There are several races of Aligote yeast, and they are all strong, with high fermentation energy. Riesling Anapsky yeast also belongs to vigorous fermenters.

For the preparation of strong wines, the Massandra 3 race is used with an ovoid cell shape, pulverized; the optimal pH value is 3.7-4.05; the optimum fermentation temperature is 18-20°C. Grape must with a sugar content of 20% is completely fermented; when fermenting concentrated grape must (30% sugar), it forms 11.8% alcohol by volume and leaves 8.7% sugar unfermented.

Race Magarach 125, named to commemorate the 125th anniversary of the first planting of grapes at the Magarach Institute, is used to produce strong and dessert wines. This race well ferments highly concentrated grape must with a sugar content of 27-30%, cold-resistant.

Race Kakhuri 2 is widely used for the preparation of champagne wine materials and wines. It ferments grape must with a sugar content of 20% with the formation of 11.4% alcohol, 0.28% sugar remains unfermented. This race is quite cold-resistant (at a temperature of 14-15 ° C, the must ferments on the 2nd day) and ferments well; the optimal pH value is 3.4-3.6.

Race Champagne 7, used for champagnization of bottled wine, is isolated from race Kakhuri 5 and is characterized by the formation of a sediment that is difficult to stir up; intensively ferments at a temperature of 4-9°C, although the wort ferments only on the 5th-6th day.

Of the wine yeasts, the Leningradskaya race is considered the most cold-resistant, and the Ashgabat 3 race is considered the most heat-resistant.

In the production of sherry, special races of yeast are used, which are a variety of the Saccharomyces oviformis species. Sherry yeast forms a film on the surface of wine in incomplete barrels, thanks to the development of which the wine acquires a special bouquet and taste.

Through careful selection of the most important production characteristics, several races of sherry yeasts (13, 15 and 20) with high film-forming ability have been identified. Later, from the production that used the Sherry 20 race, a more effective Sherry 20-C race was selected, which was widely used in many sherry factories.

In fruit and berry winemaking, selected races of yeast isolated from various fruit and berry juices are used. Fruit and berry juices are rich in yeast, which has all the qualities necessary for production and is biologically adapted to the conditions of development in the original fruit and berry juices. Therefore, yeast strains isolated from strawberry juices are used to ferment strawberry juices, and yeast strains isolated from cherry juices are used to ferment cherry juices, etc.

The following strains have become widespread in fruit and berry winemaking: apple 46, 58, cranberry 17, currant 16, lingonberry 3, 7, 10, raspberry 7/5, 25, 28, 28/10, cherry 3, 6, strawberry 7, 4 , 9.

These yeast strains ensure the normal course of fermentation, the completeness of fermentation, rapid clarification and good taste of the wine; they ferment glucose, fructose, sucrose, maltose, galactose and do not ferment lactose and mannitol.

Yeast races Moscow 30, Apple 7, Cherry 33, Chernomorodinovaya 7, Raspberry 10 and Plum 21 are successfully used in fruit and berry winemaking. Pure yeast culture Moscow 30 is recommended for fermentation of cranberry must; Apple 7 and Cherry 33 - for the fermentation of apple must; Blackcurrant 7 and Cherry 33 - for the fermentation of blackcurrant and cherry must.

4 Chemistry of alcoholic fermentation. Secondary and by-products of alcoholic fermentation
Alcoholic fermentation is a chain of enzymatic processes, the end result of which is the breakdown of hexose with the formation of alcohol and CO 2 and the delivery to the yeast cell of the energy that is necessary for the formation of new substances used for life processes, including growth and reproduction. By chemical nature, alcoholic fermentation is a catalytic process that occurs under the action of biological catalysts - enzymes.

The modern theory of alcoholic fermentation is the result of the work of many scientists from around the world.

For elucidation of the processes of fermentation, the works of outstanding Russian scientists were of great importance: Lebedev, Kostychev, Favorsky, Ivanov, Engelhardt.

According to modern concepts, alcoholic fermentation is a complex continuous process of sugar breakdown, catalyzed by various enzymes with the formation of 12 intermediate products.

1 The initial stage of glucose conversion is the reaction of its phosphorylation with the participation of the enzyme glucosinase. A phosphate residue from the ATP molecule, which is located in yeast cells, is attached to the glucose molecule, and glucose-6-phosphate is formed, and ATP is converted to ADP:

C 6 H 12 O 6 + ATP → CH 2 O (H 2 PO 3) (CHOH) 4 CHO + ADP

Glucose Glucose-6-phosphate

As a result of the addition of a phosphate residue from the ATP molecule to glucose, the reactivity of the latter increases.
2 Glucose-6-phosphate, by isomerization under the action of the enzyme glucose phosphate isomerase, is converted reversibly into the form of fructose:

CH 2 O (H 2 RO 3) (CHOH) 4 CHO → CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 OH

CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 OH + ATP →

Fructose 6-phosphate

→ CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 O (H 2 RO) + ADP

Fructose 1,6-diphosphate

Esters of glucose-6-phosphate and fructose-6-phosphate form an equilibrium mixture, called the Emden ester and consisting of 70-75% Robison ester (glucose) and 25% Neuberg ester (fructose).

The formation of fructose-1,6-diphosphate ends with the preparatory stage of alcoholic fermentation with the transfer of high-energy phosphate bonds and with the conversion of hexose into a labile oxyform, which is easily subjected to further enzymatic transformations.
4 The next most important step is desmolysis - breaking the carbon chain of fructose diphosphate with the formation of two
phosphotriosis molecules. The symmetrical arrangement of phosphoric acid residues at the ends of the fructose molecule makes it easier to break its carbon chain right in the middle. Fructose diphosphate decomposes into two trioses: phosphoglyceraldehyde and phosphodioxyacetone. The reaction is catalyzed by the enzyme aldolase and is reversible:

CH 2 O (H 2 RO 3) (CHOH) 3 COCH 2 O (H 2 RO) → CH 2 O (H 2 P0 3) COCH 2 OH +

Fructose 1,6-diphosphate Phosphodioxyacetone

CH 2 0 (H 2 ROz) SUPERB (4)

3-phosphoglyceraldehyde

The main role in further transformations during alcoholic fermentation belongs to 3-phosphoglyceraldehyde, but in the fermented liquid it is found only in small quantities. This is due to the mutual transition of the ketose to aldose isomer and back under the action of the enzyme triose phosphate isomerase (5.3.1.1)

CH 2 0 (H 2 P0 3) COCH 2 OH; £ CH 2 0 (H 2 P0 3) SWEET

Phosphodioxyacetone 3-Phosphoglyceraldehyde

As phosphoglyceraldehyde is further converted, new amounts of it are formed during the isomerization of phosphodioxyacetone.

5. The next step is the oxidation of two molecules of 3^phosphoglyceraldehyde. This reaction is catalyzed by triose phosphate dehydrogenase (1.2.1.12), whose coenzyme is NAD (nicotinamide adenine dinucleotide). The phosphoric acid of the medium is involved in the oxidation. The reaction proceeds according to the following equation: 2CH 2 0 (H 2 P0 3) SHORTLY + 2H 3 P0 4 + 2NAD Triose phosphate dehydrogenase ->

3-phosphoglyceraldehyde

->- 2CH 2 0 (H 2 P0 3) CHONCOO w (H 2 P0 3) + 2NAD H 2 (5)

1,3-diphosphoglyceric acid

The 3-phosphoglyceraldehyde molecule adds phosphate, and the hydrogen is transferred to the NAD coenzyme, which is reduced. The energy released as a result of the oxidation of 3-phosphoglyceraldehyde is accumulated in the macroergic bond of the resulting 1,3-diphosphoglycerol

6. Next, the phosphate residue of 1,3-diphosphoglyceric acid
you, containing a macroergic bond, with the participation of an enzyme
phosphoglycerate kinase (2.7.2.3) is transferred to an ADP molecule.
3-phosphoglyceric acid is formed, and ADP, acquiring
additional macroergic bond, turns into ATP:
2CH 2 0 (H 2 P0 3) CHOHCOOH co (H 2 P0 3) + 2ADP-> 2CH 2 0 (H 2 P0 3) CHOHCOOH +

1,3-diphosphoglyceric acid 3-phosphoglyceric acid

7. Then, under the action of the enzyme phosphoglyceromutase
(2.7.5.3) the phosphoric acid residue moves from the third
carbon to the second, and as a result 3-phosphoglyceric acid
lota is converted to 2-phosphoglyceric acid:

2CH 2 (H 2 P0 3) CHOHCOOH ^t 2CH 2 0HCH0 (H 2 P0 3) COOH. (7)

3-phosphoglyceric acid 2-phosphoglyceric acid

8. The next step is the dephosphorylation of 2-phospho-
foglyceric acid. At the same time, 2-phosphoglycerol acid
by the action of the enzyme enolase (4.2.1.11) by dehydro
tiation (loss of water) is converted into phosphoenolpyrovino-
gradic acid:

2CH 2 OHCHO (H 2 P0 3) COOH qt 2CH 3: CO co (H 2 P0 3) COOH + 2H 2 0. (8)

2-phosphoglyceric acid Sosphoenolpyruvic acid

During this transformation, the intramolecular energy is redistributed and most of it is accumulated in the macroergic phosphate bond.

9. Very unstable phosphoenolpyruvic acid
easily dephosphorylated, while the phosphoric acid residue
by the action of the enzyme pyruvate kinase (2.7.1.40)
together with a macroergic bond to the ADP molecule. As a result
a more stable keto form of pyruvic acid is formed
you, and ADP is converted to ATP:

2CH 2: CO syu (H 2 P0 3) COOH + 2ADP - * 2CH 3 COCOOH + 2ATP. (3)

Phosphoenol pyruvic pyruvic

acid acid

10. Pyruvic acid under the action of the enzyme pi-
ruvate decarboxylase (4.1.1.1) is decarboxylated to cleave
nii CO 2 and the formation of acetaldehyde:

2CH 3 COCOOH - * 2C0 2 + 2CH 3 CHO. (ten)

pyruvic aldehyde

11. Acetic aldehyde with the participation of the enzyme alcohol dehy-
rogenase (1.1.1.1) interacts with NAD-H 2 formed
earlier, during the oxidation of phosphoglyceraldehyde to phospho-
glyceric acid [see equation (5)]. As a result, vinegar
aldehyde is reduced to ethyl alcohol, and the coenzyme
NAD-H 2 is regenerated again (oxidized to NAD):

2SN 3 CHO + 2NAD H 2 Z 2CH 3 CH 2 OH + 2OVER. (eleven)

So, the final stage of fermentation is the reduction reaction of acetaldehyde to ethyl alcohol.

From the considered cycle of alcoholic fermentation reactions, it can be seen that 2 alcohol molecules and 2 CO 2 molecules are formed from each glucose molecule.

In the process of alcoholic fermentation, four ATP molecules are formed [see. equations (6) and (9)], but two of them are spent on phosphorylation of hexoses [see. equations (1) and (3)]. Thus, only 2 g-mol of ATP is stored.

It was previously indicated that 41.9 kJ is spent on the formation of each gram-molecule of ATP from ADP, and 83.8 kJ, respectively, goes into the energy of two ATP molecules. Therefore, during the fermentation of 1 g-mol of glucose, the yeast receives an energy of about 84 kJ. This is the biological meaning of fermentation. With the complete breakdown of glucose into CO 2 and water, 2874 kJ is released, and when 1 g-mol of glucose is oxidized to CO 2 and H 2 0, 2508 kJ is accumulated during aerobic respiration, since the resulting ethanol still retains potential energy. Thus, from an energy point of view, fermentation is an uneconomical process.

The fermentation of individual sugars occurs in a certain sequence, determined by the rate of their diffusion into the yeast cell. Glucose and fructose are the fastest fermented by yeast. However, sucrose as such disappears into the must (is inverted) at the beginning of fermentation. It is hydrolyzed by p-fructofuranosidase (3.2.1.26) of the yeast cell membrane to form hexoses (glucose and fructose), which are easily used by the cell. When there is almost no fructose and glucose left in the wort, the yeast begins to consume maltose.

The atlas of industrial alcohol yeast Saccharomyces cerevisiae race XII can serve as a reference tool for employees of distilleries providing microbiological control of production. At present, yeasts of the species Saccharomyces cerevisiae are mainly used in the industrial production of food products using yeast. In the production of bread, alcohol, wine, bread kvass, different strains (races) of yeast are used. Even the raw materials of distilleries (grain or molasses) influence the choice of one or another strain. In the production of alcohol from grain, yeast of the XII race is more often used, the permanent habitat of which is artificially prepared hydrolyzed starchy substrates. Maintaining the technology requires careful monitoring of the state of yeast and the presence of foreign microorganisms in production areas. Existing techniques make it possible to carry out the necessary microscopic analysis, but without a certain practice it is difficult to identify the obtained data of microscopic analysis and regulatory indicators of the technology.

As you know, it is yeast that converts grain substances into ethyl alcohol, and they can be considered as one of the many tools of human labor, and yeast fermentation is one of the most ancient microbiological processes used by man for his own purposes. The first mention of the use of yeast by man dates back to 6000 BC. The scientific study of yeast began in 1680 after the invention of the light microscope. Researchers from various countries have described the appearance of yeast cells; showed that yeast are living organisms; proved their role in the transformation of sugar into alcohol; obtained pure yeast cultures; classified yeast cells by mode of reproduction, nutrient intake, and appearance. Modern optical microscopes are equipped with dry and immersion objectives. An optical microscope with a dry lens allows you to study microorganisms larger than 5 microns, an immersion microscope is used to study smaller microorganisms. The invention of the electron microscope made it possible to understand the structure of the yeast cell and to study the manifestations of its genetic system, since the resolution of the electron microscope is 1.0-0.14 nm.

A microscope is an indispensable device in the production of alcohol, and without it, effective technology is impossible: it is used to determine the number of yeast cells in 1 ml of yeast or fermenting mass; percentage of budding and dead cells; the presence of foreign microorganisms; glycogen content in cells (cell fatness). The physiological state of yeast is established by the appearance of the cells, which allows the use of cheap light microscopes with dry objectives. It should be noted that the modern production of alcohol does not require a microscopic analysis of the structure of yeast cells, however, when studying the appearance of a cell under a light microscope, it is necessary to have an idea of ​​its structure.

The structure of the yeast cell

Yeast cells are round or elliptical, 2.5 to 10 µm in diameter and 4.5 to 21 µm in length. On fig. 1 is a graphical representation of a section of a yeast cell. Cell wall, cell membrane, nucleus, mitochondria, vacuoles - cell structures visible in a light microscope with a dry lens using specific dyes.

The cell wall is a rigid structure 25 nm thick, makes up about 25% of the cell's dry mass, and consists mainly of glucan, manan, chitin, and protein. The organization of the cell wall is not well understood, but current theories favor the three-layer structure model, according to which the inner glucan layer is separated from the outer manan layer by an intermediate layer with a high protein content.

The cell membrane (plasmalemma) of a yeast cell under an electron microscope looks like a three-layer structure, closely adjacent to the inner surface of the cell wall, and consists of approximately equal amounts of lipids and proteins, as well as a small amount of carbohydrates. The cell membrane acts as a permeability barrier around the contents of the cell and controls the transport of solutes into and out of the cell.

Only some progress has been made in the study of the nucleus, since individual chromosomes are very small and do not show up as discrete structures in either light or electron microscopes. Yeast cells have a single nucleus ranging in size from 2 to 20 microns. The nuclear membrane remains unchanged throughout the cell cycle. Under an electron microscope, it looks like a double membrane dotted with pores.

Mitochondria are the largest of spherical or cylindrical cellular inclusions measuring 0.2 to 2 µm in diameter and 0.5 to 7 µm in length. The two-layer shell has a thickness of about 20 nm. The number of mitochondria in a cell is more or less constant and is characteristic of a given type of microorganism.

Rice. 1. Graphic image of a section of a yeast cell (1 micrometer in 1 centimeter)

It varies depending on the stage of cell development and functional activity from 500 to 2000 mt. The functions of mitochondria are associated with the transfer of electrons, ions, and substrates within the cell. In addition, substances are synthesized in the mitochondria that accumulate the chemical energy of the cell.

Mature yeast cells contain a large vacuole. During the formation of the kidney, the vacuole, in all likelihood, breaks up into smaller vacuoles, which are distributed between the mother cell and the kidney. Subsequently, these small vacuoles merge again, forming one vacuole each in the mother and daughter cells. The function of the vacuole has not been precisely established. It contains hydrolytic enzymes, polyphosphates, lipids, metal ions, etc. The vacuole probably functions as a reservoir for storing nutrients and hydrolytic enzymes.

The intracellular content of a yeast cell (with the exception of the nucleus, mitochondria and vacuoles) is known to be called the cytoplasm, which consists of water, lipids, carbohydrates, various high and low molecular weight compounds, mineral salts, etc. An examination of the cell under an electron microscope showed a complex structure of the cytoplasm in the form granules, the functions and chemical properties of which are insufficiently studied. The cytoplasm plays an important role in the biochemistry of the cell and is in close interaction with the organelles that it surrounds.

A distinctive feature of the population of growing yeast cells is the presence of buds formed during cell division. The daughter cell arises as a small bud that grows during most of the cell cycle. Yeast growth occurs mainly during bud formation, so a bud is more or less the same size as a mature cell by the time it separates (see Figure 2). Cells may disperse shortly after dividing, but often before they diverge, new cycles of cell division begin, resulting in the formation of groups of cells. At the site of separation of cells from each other, traces remain, which are called a daughter scar in the mother cell, and a birth scar in the daughter cell. Two buds never appear at the same place on the cell wall. Each time the kidney leaves a new daughter scar on the wall of the mother cell. By the number of scars, you can determine how many kidneys a given cell has formed, which allows you to estimate the age of the cell. It has been established that haploid cells have a maximum of 18, and diploid - 32 renal scars.

Rice. 2. Graphic representation of a budding cell.

Methods of light microscopy and microbiological control used in alcohol technology.

In alcohol technology, when conducting a microscopic analysis of a yeast population with a light microscope with a dry lens, the appearance of cells is examined by the crushed drop method in unstained or stained forms (lifetime preparations), the total number of cells and the percentage of budding cells are counted, and the presence of foreign microorganisms is determined.

crushed drop method

A drop of the studied suspension with yeast cells is applied to the glass slide, which is covered with a cover glass on top. The resulting sample is viewed under a microscope, where the microorganisms are visible in different planes. This method is simple, it is used in the study of the mobility and internal structure of microorganism cells. The crushed drop method without the use of dyes makes it possible to distinguish between yeast cells by the thickness of the cell wall and membrane, the state of the cytoplasm, the presence or absence of vacuoles, the percentage of budding and dead cells, and the presence of lactic acid bacteria.

Calculation of the percentage of budding cells

To determine the number of budding cells, one drop of yeast suspension without solid inclusions and distilled water is applied to a glass slide, covered with a cover slip, excess liquid is taken with a piece of filter paper and microscoped. In mature yeast, more than 10% of the cells bud.

Example.A total of 33+35+29+32+30=159 yeast cells were found in 5 fields of vision, including budding 4+5+3+5+3=20. The percentage of budding cells is 20 x 100/159 = 12.5 (%).

Measurement of microorganism values

The unit of measure for the size of microorganisms is a micron (µm), equal to 0.001 millimeter (mm). When measuring, an eyepiece micrometer is used - a round glass with a scale applied to it (each millimeter of the scale is divided into 10 divisions). The glass is placed on the aperture of the eyepiece so that the side with divisions is at the top. To calibrate the values ​​of one division of the eyepiece micrometer, an object-micrometer is used, which is placed on the microscope stage and considered as a preparation. The micrometer object is a glass plate with a scale, one division of which is equal to 0.01 mm (or 10 microns). On fig. 3 shows the field of view of the microscope with the scales of the eyepiece-micrometer and the object of the micrometer. By coincidence of the divisions of both scales, a scale factor is set to determine the true value of one division of the eyepiece micrometer. In the figure, the divisions of the object micrometer coincided with the divisions of the eyepiece micrometer No. 2 and No. 8, or 30 divisions of the eyepiece micrometer coincided with 5 divisions of the object micrometer (comprising 50 microns). Thus, one division of the eyepiece micrometer is approximately equal to 1.67 microns (50/30=1.666...). If, instead of an object-micrometer, a preparation with live yeast is placed on the microscope stage, their visible dimensions (length and width) can be determined by examining the preparation through the same objective and eyepiece and with the same extension of the tube. To do this, it is necessary to establish to what number of ocular divisions the value of the measured object corresponds, and then multiply this number by the obtained value of the scale factor (in our case, equal to 1.67 μm). The obtained measurement results are not amenable to mathematical processing in accordance with the theory of experiment, but they give an idea of ​​the size of the studied microorganisms.

Cell Counting

To count the number of yeast cells, he uses a Goryaev counting chamber, which is a thick glass slide with transverse slits applied to it. which form three transverse

Rice. 3. Object-micrometer scales and micrometer lens for measuring the magnitude of microorganisms under a microscope

sites. The middle of them is divided into two parts, each of which is engraved with a grid (see Fig. 5) with an area of ​​9 mm 2, divided into 225 large squares with an area of ​​0.04 mm 2 each (15 rows of 15 squares) and 400 small squares with an area of ​​0.0025 mm 2 each (every third row of large squares in the horizontal and vertical direction is divided into 16 small squares). The middle platform of the glass slide is lowered by 0.1 mm relative to the other two areas, on which a special ground cover glass 18x18 mm in size is applied, which ensures the creation of a chamber for the yeast suspension. The number of cells is determined in accordance with the formula O = A x K 1 x K 2 x B, where B is the number of cells in 1 ml of suspension, pcs / ml; And the number of cells in 80 small squares, pieces; K., coefficient of chamber depth (with a chamber depth of 0.1 mm

Rice. 4. Goryaev's camera: 1 - glass slide; 2 - special cover glass; 3 - chamber for yeast suspension; 4, 6 - platform for coverslip; 5 - grid for counting yeast cells; 7 - slot for the introduction of yeast suspension

K 1 = 10; with a chamber depth of 0.2 mm K 1 = 5); K 2 - volume conversion factor, 1/ml (K 2 = 5000 1/ml); B - sample dilution factor (for yeast B=10). When counting yeast cells in a Goryaev chamber with a depth of 0.1 mm and a tenfold dilution of the yeast suspension B = 5 x 10 4 A x B.

In mature yeast and fermenting wort (during the main fermentation), the number of yeast cells exceeds 80 million pcs / ml.

Calculation of the percentage of dead cells in a yeast suspension

To determine the number of dead cells, one drop of unfiltered yeast suspension and a solution of methylene blue (1: 5000), which stains dead cells blue, are applied to a glass slide. The drop is covered with a cover glass, the excess liquid is collected with a piece of filter paper and microscopically after 2 minutes. In the field of view of the microscope, the total number of yeast cells is counted, then only blue ones, after which the preparation is moved and the count is carried out in a new field of view. Thus, the total number of cells in five fields of view is counted. After counting, the percentage of dead cells is calculated. In mature yeast, the number of dead cells should not exceed 1%. Example. A total of 43+45+39+42-40=209 yeast cells were found in five fields of vision, including blue-stained 1+0+0+0+1=2. The percentage of dead cells is 2 x 100/209 = 0.96 (%).

Rice. Fig. 5. Grid for counting yeast cells in the Goryaev chamber: 1 - large square; 2 - small square

Determination of glycogen content in yeast cells

With normal technology, glycogen accumulates in yeast when 2/3 of the must sugar is fermented and the yeast is suitable for use in production. To determine the amount of glycogen in yeast cells, a drop of unfiltered yeast suspension and 2 drops of a 0.5% iodine solution (0.5 g of iodine and 1 g of KJ per 100 ml of water) are applied to a glass slide, the drops are mixed, covered with a cover slip, taken excess liquid with a sheet of filter paper and microscope. When the ratio of yeast suspension and iodine solution is 1:2, after 2-3 minutes the cells turn light yellow, and glycogen turns brown. It is impossible to use a stronger solution of iodine than 1%, since it stains brown not only glycogen, but the entire cell. In mature yeast, glycogen occupies from 1/3 to 2/3 of the cells.

Definition of bacterial infection

To determine the percentage of bacterial infection (primarily lactic acid bacteria), one drop of yeast suspension without solid inclusions is taken from a yeast sample and placed on a glass slide, where one drop of distilled water is added. Both drops are mixed and covered with a glass slide, removing excess liquid with a sheet of filter paper, and microscoped. Since industrial yeasts are kept under non-sterile conditions by the method of naturally pure culture, a certain amount of bacteria can always be found in them. With normal technology, in sulfuric yeast in the field of view of a microscope (with an objective x40 and an eyepiece x7 or more), from 1 to 3 bacterial cells are found, among which there are usually no mobile forms. The presence of more bacteria in the field of view of the microscope indicates an increase in acidity in industrial yeast or in the fermented wort. Spore-bearing motile forms of bacteria usually do not develop during the souring of yeast mash due to the accumulation of ethyl alcohol.

Appearance of yeast cells

Pure culture dormant yeast, young, mature, old, starving and dead cells can be identified by their size and shape, structure and internal contents.

Size and shape of yeast cells

On average, the cell sizes of race XII yeast are 6x9 µm, however, depending on the environmental conditions, age and development conditions (acidity, oxygen access, etc.), their actual sizes deviate up and down. The forms of yeast of one race are determined mainly by the conditions of development. Cells are oval when cultured on grain wort; when growing on a solid medium, all yeast races produce more or less elongated cells; yeasts also have a somewhat elongated shape at the time of intensive development.

The structure and internal contents of the cell

Microscopic analysis of yeast cells should pay attention to the thickness of the membranes; type of cytoplasm; the presence of vacuoles and glycogen in the cells; number of dead cells in the population. In young cells, the thickness of the membrane is hardly noticeable, while in old cells it appears in the form of a clearly visible rim, which becomes double-contour with further aging. The type of cytoplasm can be homogeneous or granular. Granularity is mostly characteristic of old, diseased and developed under abnormal conditions (high temperature or temperature changes, high acidity, infection) cells. The lagging of the cytoplasm from the cell membrane occurs during plasmolysis or indicates the destruction of the cell. The amount of glycogen in yeast is not constant and depends on their age. The greatest amount of glycogen accumulates in mature yeast.

View of yeast cells under a microscope lens depending on their age

Appearance and contents of cells	Age of yeast cells
	Resting (pure culture)	Young (immature)	mature	overripe (old)	starving	Dead
	oval	oval	oval	Cells shrink	Cells cringe
The size			Large	Decreased in size	Decreased in size
budding cells	No or single	Budding 10%	Budding 10%	No or single
Shell	Very thin	Very thin	well-defined	Thick or double-sided	Thick or double-sided	Dissolves and disintegrates
Cytoplasm	homogeneous	Soft and uniform	Heterogeneous or grainy	very grainy	very grainy	Lumpy
Vacuoles				Sometimes occupies the whole cell
Glycogen	in single cells	Takes less 1/4 cell or missing	Occupies from 1/3 to 2/3 of a cell	In small quantities	Missing	Missing

Type of yeast cells depending on age

In young yeast the membrane is very thin, the cytoplasm is tender and homogeneous. There are no vacuoles or small vacuoles are visible in a small number of cells. Glycogen in single cells. mature yeast have well-defined shells. Noticeably 10-15% of cells with kidneys. Heterogeneity, granularity is visible in the cytoplasm, medium-sized vacuoles appear, the cells contain a lot of glycogen. The number of dead cells does not exceed 1%. At overripe yeast a thick shell is clearly visible with a strong granularity of the cytoplasm. Large vacuoles occupy almost the entire cell. If the yeast lacks nutrients, then the cells decrease in size. Single cells bud. The percentage of dead cells progressively increases with aging.

Shells starving yeast thick (in some cells, the membranes have a variable thickness), their contents are granular. Cells decrease in size, shrink, slightly elongate. There are no vacuoles, no glycogen. Death and destruction of yeast takes place in several stages. The cytoplasm becomes lumpy, but adheres to a well-visible membrane. Then the shell blurs and disintegrates. The protoplasm becomes even more granular and breaks up into small pieces. Sometimes the shell remains, but the protoplasm lags behind it, gathers in a lump in the center, the cell lengthens, takes an irregular shape and collapses. The table shows data on the appearance of yeast cells depending on their age.

Appearance of yeast cells during yeast generation

At the start of the plant (during the development of production, at the beginning of the season or when the equipment is infected), yeast is prepared from a pure culture that enters the plant in a test tube. Breeding a pure culture is carried out by successive transfer of cells from a test tube to a 500 ml flask, then into a five-liter bottle and mother liquor, from where the yeast enters the yeast, where production yeast is prepared.

Pure yeast culture

On fig. Figure 6 shows an image of the field of view of a microscope with yeast cells transferred from a test tube with a pure culture to a flask with wort. Cell membranes are very thin, the cytoplasm is tender and homogeneous, there are no vacuoles. There are no lactic acid bacteria in the field of view of the microscope, which indicates the good quality of the pure yeast culture. On fig. 7 Yeast from a 500 ml flask after 24 h of growth. Thin shells, homogeneous cytoplasm of cells and the absence of vacuoles in it indicate the youth of yeast. The absence of lactic acid bacteria in the field of view of the microscope and a large number of dividing cells (more than 15%) once again confirm the good quality of the pure culture.

Production yeast

The quality of yeast before being transferred to production is determined by the number of budding cells, the presence of lactic acid bacteria in the yeast, the number of dead cells, the fatness of the yeast (the amount of glycogen in the cells), the number of cells in 1 ml of yeast. On fig. Figures 8-11 show images of the fields of view of a microscope with samples of mature yeast from one yeast when determining their quality before transferring them to production.

All images show large oval-shaped cells with clearly defined membranes and granular cytoplasm. More than 10% of the cells bud, and in the field of view of the microscope there are no more than 3 cells of lactic acid bacteria (see Fig. 8). The number of dead cells does not exceed 1% (see Fig. 9). The content of glycogen indicates the fatness of the yeast (see Fig. 10). The number of yeast cells is 120 million pieces/ml (see Fig.-11). Based on the analysis carried out, only one conclusion can be drawn: the yeast in yeast is of good quality and can be transferred to production.

In some cases, yeast infection occurs, primarily with lactic acid bacteria. On fig. 12 is an image of the field of view of a microscope with samples of mature infected yeast. Large oval cells with well-defined membranes and granular cytoplasm. A significant number of cells bud, but there are more than 3 cells of lactic acid bacteria in the field of view of the microscope. Such yeast is not suitable for use in production.

When distilleries stop (lack of sales of finished products or overhaul), yeast is stored at a temperature of 10 ... 12 ° C for several months. On fig. 13 shows an image of the field of view of a microscope with a sample of chilled yeast from yeast, which was stored at a temperature of 7 ... 10 ° C for 45 days. Yeast cells vary in size and shape. Some cells have an oval shape and racing membranes with a homogeneous cytoplasm, like young or mature cells. Other cells have lost their shape, thick membranes of variable thickness, the cytoplasm is highly granular, which allows them to be attributed to starving and overripe cells. Chilled yeast is used in production. On fig. 14 shows an image of the field of view of a microscope with a sample of mature yeast from yeast, in the cultivation of which cold yeast was used. The cells are large, oval in shape, with clearly defined membranes and granular cytoplasm. Some cells bud, the number of lactic acid bacteria cells does not exceed the norm. Two cells have destroyed shells. In all likelihood, these are the remains of cold yeast cells. The yeast is suitable for use in production.

Rice. 6. Pure yeast culture

Rice. 7. Pure yeast culture after 1 day

Rice. 8. Mature yeast from yeast

Rice. 9. Mature yeast (calculation of the percentage of dead cells)

Rice. 10. Mature yeast (determination of yeast bodyness)

Rice. 11. Mature yeast (counting the number of cells in one milliliter of yeast)

Rice. 12. Mature infected yeast

Rice. 13. Mature yeast from yeast after 45 days of storage at a temperature 7.. .12 °С

Rice. 14. Mature yeast from yeast grown from chilled yeast

Appearance of yeast cells during wort fermentation

When fermenting the wort, it is advisable to conduct a microscopic analysis in the case of an increase in the titratable acidity of the mash during fermentation by more than 0.2 °K (souring of the mash). On fig. 15 shows a microscope view of a sample from a soured fermentation tank (periodic wort fermentation scheme, 72 hours of fermentation). Since the fermentation of the wort is over, the analysis of the appearance and internal contents of the yeast cells does not give a result. A large number of lactic acid bacteria in the field of view of the microscope indicates bacterial souring of the fermentation tank.

Rice. 15. Infected fermentation tank brew

Currently, distilleries use several technological schemes for the production of alcohol from grain, which differ in the temperature of the heat treatment of raw materials: using apparatuses of the "Genz" type - up to 165 ° C; units of continuous cooking (Michurin scheme) - up to 150 °C; devices for hydrodynamic processing of the batch - up to 95 °C. In addition, distilleries use various saccharifying materials: malt; crude enzyme preparations obtained in the conditions of an alcohol plant; purified enzyme preparations produced by specialized biochemical plants. The methods of heat treatment of the batch and the enzyme preparations used affect all technological indicators, including the indicators of yeast preparation and wort fermentation. The atlas provides recommendations on the use of microscopic analysis in the production of alcohol from grain using devices for hydrodynamic processing of the batch, purified enzyme preparations and sulfate yeast.

Pure Yeast Culture Infection

Microscopic analysis of a yeast sample from a test tube with a pure culture or a flask after 20 hours of growth showed the presence of lactic acid bacteria in the microscope fields. A pure yeast culture is infected (as a rule, this occurs during long-term storage at high temperatures). It is necessary to change the pure yeast culture. If infection is re-identified in a pure culture, it is advisable to change the supplier of the pure yeast culture.

Industrial Yeast Infection

Microscopic analysis of a sample of mature yeast from yeast showed the presence of more than 3 cells of lactic acid bacteria in the field of view of the microscope, which indicates infection of mature yeast. Yeast infection occurs as a result of the following main reasons: the use of low-quality grain; the use of water from open reservoirs (especially in the warm season); the use of low-quality enzyme preparations; poor-quality washing and sterilization of equipment and pipelines; violations of regulatory indicators for the preparation of yeast; operation of obsolete equipment at the plant.

In the cost of alcohol, the cost of grain takes 40-60% and the use of cheap grain improves the economic performance of production. However, when using low-quality raw materials, alcohol losses occur as a result of infection. It is advisable to use grain with a quality not lower than the first degree of defectiveness: grain that has left the dormant stage; showing enhanced physiological processes (respiration) that contribute to the vital activity of microorganisms; having malty or putrid odors, but suitable for production. If it is necessary to process low-quality grain, the temperature of the heat treatment of the batch should be increased to 130...135 °C.

When using water from open reservoirs in the warm season, the temperature of the heat treatment of the batch can be increased to 130...135 °C. It is preferable to use drinking quality water from a water supply or artesian well. It is advisable to use methods for disinfecting water or batches by treating them with magnetic and other radiations used in the food and medical industries in the processing of food and medical equipment.

If it is not possible to find the source of infection of mature yeast, then the enzyme preparations are checked for their bacterial contamination. Enzymes are the first to be infected. produced in the conditions of distilleries and unrefined (in liquid form) transported by road or rail (especially in the hot season). When enzyme preparations are infected, they are replaced with high-quality ones and the supplier of enzymes is changed.

Equipment washing during yeast generation is carried out with brushes and water from hoses (pressure 3-4 kg/cm 2 ) followed by steam sterilization. Steam consumption is 10-12 kg per 1 m of yeast with 30-minute steaming. Washing of pipelines is carried out with various washing solutions, followed by steam sterilization. The most difficult to clean and sterilize internal coils. It is advisable to replace the yeast cooling coils with cooling jackets, and wash the inner surface with warm water at a pressure of 120-150 kt/cm: using high-pressure cleaners. The greatest effect from the use of such cleaners is achieved when washing butt and fillet welds inside the equipment, as well as when washing the inner surface of yeasts with corrosive shells. The use of cleaners makes it possible to reduce the consumption of steam and cleaning solutions, as well as eliminate manual labor when cleaning the internal surfaces of the equipment with brushes.

Washing and sterilization of pipelines is carried out in accordance with the regulations. The most difficult is washing and sterilization of heat exchangers of the "pipe in pipe" type, which cool the saccharified mass from 52 ... 60 ° C (depending on the enzymes used) to 22 ... 28 ° C (depending on the yeast used), especially if often there is a stop of the pumps pumping the batch into the saccharifier, which leads to a delay in the mass in the heat exchanger. It is expedient to replace the tube-in-pipe heat exchanger with a plate heat exchanger, which is ten times smaller in size, made of stainless steel and easy to clean and sterilize when disassembled.

When preparing yeast, it is necessary to adhere to the indicators of the technological regulations. The most difficult thing is to ensure that enough water is supplied to the yeast coils (especially in the warm season) and to transfer mature yeast to the fermentation tank without delay. Replacing the cooling coils with a cooling jacket makes it possible to increase the cooling surface of the yeast by several times and, in the absence of cold water, to achieve cooling of the yeast mass to the required temperature. Having a significant cooling surface in yeast, it is possible to achieve timely supply of yeast to the fermentation tank by changing the temperature of yeast generation. Reducing the temperature of yeast generation to 25...27 °C provides an increase in the time of yeast preparation, and an increase in the temperature of yeast generation to 30...32 °C speeds up the preparation of yeast.

In the technology of alcohol, capacitive equipment is usually made of black steel with a wall thickness of 5-8 mm. The large wall thickness allows the use of yeast and pipelines up to 25 years without repair. During this long time, shells form on the walls of the yeast for various reasons (metal corrosion, cavitation processes in the liquid, metal fatigue), which are poorly washed and contribute to the infection of mature yeast. It is necessary to change the equipment in time (once every 6-7 years of operation) and, thereby, exclude foci of yeast infection.

Insufficient nutrition of yeast cells

Microscopic analysis of a sample of mature yeast from yeast showed that glycogen in the cells occupies less than 1/4 of the internal content, and the yeast cells decreased in size. This indicates that the yeast is either not ripe and it is too early to transfer it to production, or it has stood and the cells need additional nutrition. In the first case, it is enough to increase the yeast generation time. In the second, it is advisable to check the duration of the hydrodynamic treatment of the grain batch (the completeness of filling the apparatus for the hydrodynamic processing of the batch in accordance with the regulations), which determines the amount of soluble solids of the raw material and, in particular, the dissolution of grain proteins, since the lack of nitrogen nutrition reduces the fermentation activity of yeast; correct dosing of enzymes in the saccharifier. With a lack of nitrogenous nutrition, it is possible to use carbamide, which is taken into account and dosed based on the nitrogen content in it.

Increased number of dead cells

Microscopic analysis of a sample of mature yeast revealed that the content of dead cells exceeds 1% of the total number of yeast. Excessive death of yeast cells occurs when the temperature rises during yeast generation above the regulated value (30 °C) or when the acidity of the yeast wort increases (above 1.1 °K). It is advisable to monitor the implementation of regulatory indicators of yeast generation.

Reduced number of cells per 1 ml of yeast and insufficient number of budding cells

Counting the number of yeast cells under a microscope showed that their content in yeast is 80 million pcs / ml, and counting the number of budding cells revealed that less than 10% of budding yeast in the field of view of the microscope. It is necessary to check the fulfillment of all regulatory indicators, the quality of grain, enzymes, sulfuric acid (determine the presence of arsenic in it). Substandard raw materials and auxiliary materials should be replaced.

Fermented wort infection

Microscopic analysis of a sample of the fermented wort showed the presence of a large number of lactic acid bacteria. A decrease in the yield of alcohol from 1 ton of grain should be expected, since the nutrients of the raw materials are processed by bacteria into lactic acid. The reasons for the infection of the mash can be: violation of the regulatory parameters during fermentation; unreasonable increase in the time of fermentation of the wort, when the amount of unfermented carbohydrates in the mash is less than 0.65 g/100 ml (with hydrodynamic processing of the batch after 48-60 hours of fermentation), and the mash continues to be aged in the fermentation tank for up to 72 hours; lack of cooling water.

In case of violation of the regulatory indicators of wort fermentation and an unreasonable increase in fermentation time, it is sufficient to carry out organizational measures that ensure technological discipline at the enterprise. If there is a lack of cooling water, technical measures must be taken. The use of cooling jackets instead of coils makes it possible to increase the cooling surface of fermentation tanks several times, which significantly reduces water consumption. At plants that use remote heat exchangers of the “pipe in pipe” type for cooling the mash, it is advisable to replace them with plate heat exchangers, which will allow more efficient cooling of the mash without changing the temperature of the cooling water. Cooling water deficiencies can be compensated by lowering its temperature, through the introduction of cooling towers and refrigeration units.

CONCLUSION

In the production of alcohol, the main component of the technology is yeast, which requires great attention and responsible attitude of the attendants, which is possible only with the help of microscopic analysis of both individual cells and the yeast population as a whole. By the appearance of the cells, it is possible to determine the physiological state of the yeast and make adjustments to the technology. The authors believe that the microscopic images of yeast presented in this atlas will facilitate the work of distillery personnel in breeding pure yeast culture, yeast generation and wort fermentation.

Literature

1. GU 9182-160-00008064-98. Pure yeast culture. Race XII.

2. Pavlovich S.A. Medical microbiology. -Minsk: Higher school, 1997. 133 p.

3. Yarovenko and others. alcohol technology. -M.: Kolos, 1996. 464 p.

4. Ternovsky N^S. and etc. Resource-saving technology in the production of alcohol. -M.: Food industry, 1994. 168 p.

5. Sasson A. Biotechnology: Accomplishments and Hopes. -M.: Mir, 1987. 411 p.

6. Rukhlyadeva A.P. and etc. Instructions for technochemical and microbiological control of alcohol production. -M.: Agropromizdat, 1986. 399s.

7. Bachurin P.Ya., Ustinnikov B.A. Equipment for the production of alcohol and alcohol products. -M.: Agropromizdat, 1985. 344 p.

8. Berry D. Biology of yeast. -M.: Mir, 1985. 95 p.

9. Konovalov S.A. Biochemistry of yeast. -M.: Food industry, 1980. 272 ​​p.

10. Seliber G.L. Large workshop on microbiology. -M.: Higher school, 1962. 420 p.

Alcoholic fermentation- the foundation and the beginning of all drinks containing alcohol, whether it be wine, whiskey or beer. The basis of this very foundation is raw materials, water and yeast. In this article, we will cover the different types of wine yeast used in home and industrial winemaking. Let's consider what kind of yeast are - friendly, helping to develop the richness and diversity of wines, and hostile to the winemaker, oppressing and spoiling not only the wine itself, but also infecting entire wineries along with the equipment.

Alcoholic fermentation (aka “fermentation”) is a biochemical process carried out by yeast, the ideal result of which is the conversion of saccharides (mainly sucrose, glucose and fructose) into ethyl alcohol (the main product), carbon dioxide and many chemical trace elements (necessary and not useful, harmful, and beneficial by-products).

Yeast- microscopic unicellular fungi. Modern microbiology divides them into more than one and a half thousand species and another thousand subspecies, and they, in turn, can reproduce many variations - depending on the results of controlled and uncontrolled mutations and rebirths (you must have come across this yourself if you used one and the same yeast several times, propagating them independently).

Since ancient times, man has adopted alcoholic fermentation, but the food industry and science are still discovering more and more new possibilities and features of using yeast to produce ethyl alcohol. A lot of efforts are concentrated precisely in the development of the winemaking market and the microbiology associated with it, this is a whole industry - oenology. Oenology is engaged in the study and breeding of bacteria, the development of enzymes, the research and reproduction of yeasts that have the qualities necessary for winemakers, allowing the production of many wines and wine drinks, discovering new facets and tastes, as well as preserving old and rare ones that have become the historical heritage of mankind.

The main types of yeast (saccharomyces - they are the friends of all alcovars - winemakers, brewers and moonshiners) used in the production of alcoholic beverages (including at home).

Table for clarity. Here are some races of yeast, variations of which (different strains of the same species) are popular in industrial (and some in home) winemaking. Please note that the choice of a particular race is determined, among other things, by the conditions of fermentation. Some races of wine yeast recommended for winemaking are shown in the table (some of their foreign analogues are available for purchase in our store).

"Saccharomyces cerevisiae"(Saccharomyces cerevisia) is the most common, diverse and "tamed" type of yeast currently in the world. It is the various races of this type of Saccharomyces that are leaders in the field of bakery, wine, beer and alcohol yeast. They are so diverse, and their scope is wide, that they deserve a separate article. Unfortunately, they are not always encountered in wild winemaking, and considering that among Saccharomycetes cerevisia there are hundreds of subspecies negatively (to one degree or another) affecting wine, the probability of "successful infection" becomes even lower, but not absent;

"Saccharomyces vini"(Saccharomyces blame) - they mainly live on ripe (and especially on damaged) grapes and in juices (uncovered from external influences). Often they can be found in the soil, in the digestive system of insects (especially fruit flies, wasps and bees), as well as inside the wine industry (including home wineries) - on walls, utensils and equipment. However, in home winemaking they are used very limitedly, they can have many undesirable effects - for example, clouding of the wine and the formation of suspension;

"Saccharomyces oviformis"(Saccharomyces oviformis) - most often the use of this race in winemaking can be beneficial. They are used to ferment musts with a high sugar content and are well suited for the production of dry wines. Modern representatives of this yeast race are popular in the production of champagnes.

There are also domestic races: “Leningrad”, “Kyiv”. The disadvantages of using these strains may include re-fermentation in the finished wine (most often semi-sweet, but not exclusively), as well as turbidity and the formation of late sediment. The most productive use of these strains for the production of Sherry - fortified wine. Representatives sharpened for this (a variety called “ Oviformis Cheresiensis”) - “Sherry 96-K” and “Sherry-20-C” - however, they very quickly generate a film on strong wine (16-17% vol.).

"Saccharomyces bayanus (uvarum)"(Saccharomyces bayanus uvarum) - most often they can be found in fruit wines and juices. This is a very leisurely yeast - it develops slowly, the mutation is difficult to control due to the microbiological processes that are difficult to distinguish for the current level of control in home winemaking. They are not yeasts with a high degree of attenuation (alcohol formation), but they have a rare feature - increased stability and resistance to cold. As for the products of fermentation, they are almost identical to what the aforementioned yeast strain produces. S. Vini. Features - many (but not all) races of this variety are able to form the densest (not amenable to resuspension) yeast sediment, the foam is almost completely absent, they give an increased content of glycerin. Of the most popular races - "Novotsimlyanskaya 3", while it is almost inaccessible for home winemaking, but has proven itself well for the production of semi-sweet wines.

But not all yeasts are the same. Next, consider a few dangerous and vile enemies of winemakers - yeast, which has completely unfriendly properties.

"Pichia"/"Hansenula"/"Candida" and other filmy are serious enemies, pests and culprits of failed wines (especially when using wild yeasts). Their main feature is the formation of a film on the surface of the wine, especially under aerobic conditions ( water seal (water seal) to help). The cells of these harmful yeasts have an unstable shape - there are elliptical, oval, sausage-shaped and club-shaped forms and disproportionately elongated. Some of them (Pichia and Hansenula) form spores, while others reproduce by budding. These races are capable of fermenting wine must at high speed, oxidizing it. Some of them cannot produce enough alcohol for modern wine, for example, Hansenula - gives only up to 5% ethanol.

In a properly prepared wort, they are usually not dangerous, because. are contained in small quantities. If you comply with the technological conditions for the preparation and storage of wine materials (tightness, sterility of the material and the surrounding atmosphere), from which the wine will be made, there is nothing to fear. But with poor preparation (beginner/ignorant winemakers like not to wash the grapes (not knowing its microflora), use unprepared water and containers) - the strain quickly begins to multiply on the surface. This leads to the creation already for 2-3 days (sometimes later) of a glossy, and then a folded (pimply) film, which can be in a wide color range - from white and dull-transparent to gray.

If the wine is suddenly infected, then the film is not the worst. Film formation signifies a new stage of fermentation - fermentation with sugar oxidation. In the process, not only ethyl, but amyl and butyl alcohols, as well as acetic, butyric, succinic acids and various ester compounds of these acids are necessarily formed. As a result, the wort gets a characteristic very unpleasant odor. The probability is especially high if you use pulp - then the most favorable conditions come for races like Pichia / Hansenula (therefore, we recommend using stronger fruit and alcohol yeast for the production of chacha, which can level this process).

Some filmy yeasts (often found in wild fermentation - can be conversely very alcohol-resistant (and also sulfite-resistant), which some unlucky winemakers like at first. They can feel comfortable even at an alcohol content of 14% vol. and 400-500 mg / l SO2. For the same reason, in table wines (to which there is access to oxygen) they will feel very comfortable, gradually forming various impurities that lead to deterioration of wine (especially homemade and unfinished wine without preservative). They restore very quickly sulfates and sulfites, while generating a high content of hydrogen sulfide and various sulfur compounds, this is the cause of an unpleasant rotten smell.

Blossom is one of the most common wine diseases that gives an unpleasant appearance (precipitation, suspension, islets, haze) and an unpleasant smell, often due to the presence of active yeast of the aforementioned races and breeds in the must. In addition, they are the worst enemies of the production of dry wines, champagne and sherry - be careful!

The development and reproduction of malignant yeast in wine can be prevented by limiting the access of oxygen to it (again, a water seal) and by setting the right conditions for storing wine material - low temperatures, and the containers themselves must be completely filled - without leaving a large amount of free space. Sometimes timely topping up may be required - do not neglect this.

"Zygosaccharomyces"- another representative of the harmful (for the winemaker) microflora. This race of yeast is very osmophilic - they feel great in extremetea-sweet wort, develop even with a sugar content of 60 and even 80 grams per 100 ml. (vacuum fermentation of the wort, various homemade preparations - honey, jams, preserves). They cause the fermentation process in such extreme conditions (imagine what an incredible hydromodule it is) and thereby drastically spoil the product. Paradoxically, this strain is not very alcohol-resistant either - they form on average no more than 10-11% alcohol, while they ferment for an extremely long time, but during this time they manage to completely spoil the once suitable material / product. However, modern science has also found their use, though limited - "Race Vierul, Maikopskaya, Krasnodarskaya 40" - can be used to reduce acidity in a very acidic wort, because. process the material mainly by fermentation, not oxidation (like other harmful ones).

"Saccharomy codes"- regular pests of ancient and modern winemaking. These are complex yeasts that have a large form and an unstable type of reproduction - they can both divide and bud. They have an alcohol resistance of up to 12% (and according to some reports up to 14%), and the wort itself is enriched with ethyl acetate, which has a detrimental effect on the survival of pure yeast cultures and can cause various side effects (for example, a sudden resumption of fermentation).

Saccharomycodes ludwigii- can cause the resumption of fermentation even highly sulphated wort (not all preservatives are a panacea). They also withstand very high SO2 content in finished wines and musts.

"Hanseniaspora"(apiculatus) - an extremely common yeast that can be represented as both sporogens (Hanseniaspora apiculata) and asporogenic fungi (Klocker apiculata). If the fruit is damaged, it is very likely that they are already there, do not disdain juices and large fruits (and small ones too, just a little less often). Fun fact: when grapes ripen, they can reach up to 99% of all the yeast that is there (another reason to be clean - wash the grapes and all wine material!). The fermenting ability is low - only about 4-7% vol. alcohol, however, volatile compounds, ethyl acetate, butyric, propionic and other acids accumulate a lot. Often they are the reason that the must or wine (especially homemade) is not fermented. They have explosive growth and multiply very quickly in the must, several times faster than the growth of other yeasts - especially noble ones. They give the wine bitterness, a lot of extraneous and at the same time strong odors, as well as acetic shades. Did you make homemade champagne or sherry and got “sticky” leftovers? This is also their work. Sulfitation (sometimes increased) and long-term settling can help.

"Torulopsis"- a common race harmful to winemaking, especially when it comes to grapes. They actively stand out in the already fermented grape juice, which began with wild yeast (the so-called noble rot). They can be divided into two main species (T. bacillaris and T. candida). In recent years, there has been information that they form spores, but at the moment it is generally accepted that they reproduce by budding. They do not often appear in wine, but a frequent guest in grape juice. Capable of fermenting wort up to 12.5% ​​ethanol turnover. In terms of biochemistry, they are not as harmful and destructive as other yeasts that manifest themselves during wild fermentation, they form much less harmful chemical elements, but they form - mucus. Agree, few people will be pleased to drink wine, in which in some places there are islands resembling jelly or something even more slippery. They are also osmophiles (they feel good even if the sugar content is 60-80 grams per 100 ml) and love high temperatures, and the increase in SO2 is not at all noticeable for them.

Rhodotorula- this is the so-called "pink yeast", they are so called because of the characteristic color. They do not ferment sugars, but oxidize them, thus creating a dense pink film. They contribute to the oxidation of juices, the formation of turbidity and the precipitation of dessert and semi-sweet (well, sweet) wines. An interesting feature is that they can feed on alcohol vapors in the air, for this reason they often find themselves “red-handed” on the walls of wineries and even cellars in the form of pink slime.

Instead of output:

Under industrial conditions, spontaneous fermentation can cause undesirable consequences. To avoid this and obtain good quality wine, fermentation is carried out on pure cultures of specially selected yeast races, introducing them into the must for a directed process. In modern winemaking, it is still quite common to stumble upon neutral and suitable yeast for winemaking, they may not be true (ideal, like pure yeast cultures), but they will not spoil the wine. For this reason, it cannot be unequivocally assumed that spontaneous fermentation will necessarily lead to spoilage of wine, but this possibility always exists and sometimes this probability can categorically increase. For example, if harmful killer yeast is found on one bunch of grapes, during fermentation they are able to destroy sensitive ones with great speed (1 killer cell can kill an average of 20 noble cells). In addition, there are evilly neutral (not participating in intraspecific struggle) races that can saturate the wine with unnecessary chemical compounds, usually smelling bad.

The use of wild yeast is often fraught with unkindness - suddenly the wort stops "boiling" or a "different fermentation" (filmy) begins. Therefore, if you categorically use wild yeast - add sulfur dioxide , noble strains are usually resistant to sulfitation, and harmful yeasts are usually not (but, unfortunately, not all)

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