Purpose of the saccharification process

The liquefaction and saccharification of starch by enzymatic hydrolysis is well researched and studied. Its purpose is to convert the starch contained in the boiled mass into sugars (maltose + dextrins) under the influence of green malt amylase or moldy mushrooms in preparing it (starch) for fermentation.

In 1811, an associate of the Russian Academy of Sciences, Konstantin Kirchhoff, discovered the transformation of starch into sugar when boiled with sulfuric acid. For this discovery, he was elected an extraordinary academician and awarded a pension. In 1814, Kirchhoff discovered another equally important catalytic reaction - the action of malt diastase on starch.

In the article "On the preparation of sugar from starch," Kirchhoff pointed out that "the high price of Arabic gum prompted me to look for a cheap surrogate for the latter. And it seemed to me possible and achievable to eliminate the gelatinous state of boiled starch by means of dilute mineral acids and heat, and if this would be possible, I assumed I, then it (starch) should have looked like Arabic gummi". Indeed, today it is well known that sulfuric, nitric and oxalic acids destroy the gelatinous state of starch and under their influence, with prolonged heating, starch is converted into glucose.

To study the evolution of ideas about the process of hydrolysis, a special case of which is the saccharification of starch, the views of Professor A.N. Khodnev.

In 1852, Professor Khodnev suggested that a catalyst is a chemically active substance that gives intermediate products. Professor Khodnev explained the catalytic effect of acids on starch and its conversion into glucose by the preliminary formation of "pair compounds", for example, sulfuric acid is attached to starch, and this compound easily decomposes when heated with water into sulfuric acid and carbohydrate, which absorbs water and turns into grape sugar.

The action of green soda diastase on starch, according to Professor Khodnev, also consists in the gradual formation and decomposition of "paired compounds".

Recently, the nature and composition of enzymes have become known. It has been established that the enzyme consists of a protein part (apoenzyme) and a protein-free part (prosthetic), called the coenzyme.

The coenzyme can be separated from the apoenzyme by dialysis, and in the free state, the coenzymes are thermostable. When a coenzyme is combined with an apoenzyme, the activity inherent in the enzyme molecule is restored.

The apoenzyme molecule apparently has the functions of activating polar groups and binding the enzyme to the substrate.

The connection of the enzyme with the substrate can be inhibited by substances that form stable compounds with the enzyme.

The assumption about the formation of intermediate compounds between the enzyme and the substrate was previously based mainly on the study of the kinetics of reactions under various conditions. At present, the formation of complexes with the substrate by peroxidase and catalase has been proven by spectrophotometric analysis.

In close contact of the reactive group of the enzyme with the reactive group of the substrate, an enzyme-substrate complex is formed.

In the enzyme-substrate complex, there is a bond between the polar groups of the enzyme and the substrate.

The binding mechanism of the enzyme-substrate complex has also been proven using glucose phosphates specially labeled with C 14 atoms.

The connection of the enzyme with the substrate depends on the spatial arrangement of the reacting groups of the enzyme and substrate and their configuration.

Many details of the mechanism of formation of the enzyme-substrate complex have not yet been sufficiently studied, but it can be said with certainty that several reaction groups of the substrate and the enzyme are involved in its formation. This position is confirmed by the specificity of enzymatic reactions, and the shape of the surfaces of the reacting groups of the enzyme and substrate plays an important role in this.

As is known, the enzymatic hydrolysis of starch under the conditions of alcohol production produces maltose and a mixture of intermediate products called dextrins.

Maltose is easily fermented by yeast to form alcohol (and fermentation by-products) and carbon dioxide, while dextrins are converted to sugars and fermented during the post-fermentation period under the action of diluting amylolytic enzymes.

The process of starch saccharification proceeds in two stages: in the first there is a decrease in the viscosity of the starch solution (liquefaction) and in the second - the actual saccharification (conversion into sugars and dextrins).

An enzyme or other catalyst changes the reaction so that it is possible at a lower activation energy. Thus, the inversion of sucrose requires an expenditure of 26,000 cal/mol, and under the action of the enzyme only 13000 cal/mol. Due to the decrease in the activation energy, the reactions proceed at a faster rate, since most of the molecules become sufficiently active.

The activation mechanism can be seen as a result of the collision of reacting molecules or an increase in collisions within molecules.

As a result of the chemical and adsorption interaction of the enzyme with the substrate, an intermediate complex is formed, the rate of decomposition of which determines the rate of this reaction. For example:

The reaction rate can be determined by the number of active molecules, i.e. molecules that have sufficient activation energy and react per unit time.

During enzymatic processes, the equilibrium constant does not change, only the rate of the reaction in one direction increases.

The transition of malt amylase into solution can be accelerated by creating conditions that favor the osmotic penetration of water into the germinated malt, followed by diffusion of the amylase through the walls of the malted grain.

A decrease in amylase activity under the action of certain additives is associated with the adsorption of certain substances at the site of their active groups. Amylase, which has active groups, is capable of adsorbing inorganic and organic substances.

Blocking the active groups of amylase of metals, such as iron, aluminum, lead, when the salts of the corresponding metals are dissolved, leads to the fact that the polar groups cannot exercise their functions, i.e., actively interact with the polar groups of starch.

Zabrodsky and Vitkovskaya showed that melanondin substances have an inactivating effect on the amylolytic enzymes of malt, and established their negative role in the process of saccharification of boiled mass starch.

A portion of the dispersed raw material (50 or 100 g) was transferred into a liter flask and water was added in a ratio of 1:2.5.

The mixture was thoroughly stirred with a stirrer (from an electric motor) at room temperature for 30-40 minutes, after which it was heated to 55° and saccharified for 30 minutes with a malt extract. A 20% malt extract was prepared from equal parts of barley and millet malts.

The extract was added to the saccharified dispersed grain raw material at the rate of 16% malted grain (barley and millet) in relation to raw starch.

Under the action of a-amylase and b-amylase on amylopectin, an unsplit residue containing phosphodextrins remains. Breaking bonds with phosphoric acid is achieved by the action of a dextrinolitic enzyme - dextrinophosphase, abbreviated as dextrinase. Therefore, for the complete breakdown of the starch molecule, the presence of dextrinase.

The increase in a-amylase activity has a slightly different character. In dormant barley grains, a-amylase activity is zero, and only after long-term storage in the grain can traces of it be detected. When grain is germinated on the third or fourth day, a jump in the increase in the content of a-amylase is observed, after which a-amylase activity gradually increases. At a temperature of 12-14 C, the limit is reached in 11-14 days, at a temperature of 18-20 C on the seventh day, and at a temperature of 27-28 C on the fifth day.

Dextrinase, like amylase, accumulates as the grain germinates. At the beginning of germination, the accumulation of dextrinase, like all grain enzymes, occurs slowly, then, after four days, faster, and at the end (on the tenth day) it almost stops. The figure shows a graphical representation of the dynamics of accumulation of amylase and dextrinase under the conditions of current malting for barley, oats and millet.

The duration of germination is closely related to temperature, the lower the temperature, the longer it takes to germinate the grain.

The malt of different cereals contains different amounts of these enzymes. Thus, four groups of cereals are distinguished:

One grain crop is not enough to grow for malt. Take 2.3 malt to get a high content of all enzymes. Most often they take barley and rye malt (sources of alpha and beta-amylase) and millet malt (dextrinase). Or the sum of three malts: barley, millet and oat.

At domestic distilleries, undried malt is used for saccharification. It can not be stored for a long time, so every alcohol. the plant prepares it in the amount necessary for the current work.

The end products of starch saccharification under the action of malt amylase are maltose and dextrins. The ratio between the amount of these products and the malt amylase acting on the starch is not constant and depends on many factors, mainly on the saccharification temperature.

Pronin showed that with an increase in the amount of malt amylase, the final ratio between maltose and dextrins changes to a very large extent towards maltose. The question arises about the optimal amount of malt required for saccharification.

Malchenko and Krishtul, studying the saccharification of starch with different amounts of malt, showed that for saccharification it is possible to use a smaller amount of malt compared to that accepted in industry - up to 5% by weight of the processed raw materials.

They established the optimal amounts of malt required for saccharification of boiled starch-containing raw materials. To study the process of saccharification of dispersed raw materials and determine the optimal amount of malt, we studied the kinetics of saccharification of dispersed raw materials with malt amylase.

For these studies, we took 50 G dispersed oats and 150 ml water. The suspension of dispersed oatmeal was stirred with a stirrer at room temperature for 30 minutes, after which the flask was heated to 57° and kept in a water bath at 59°.

The given data show that the optimal amount of malt required for the saccharification of dispersed starch-containing raw materials lies in the range of 6-8% by weight of the raw materials to be saccharified, which was also confirmed by the fermentation of dispersed oats.

We conducted all factory studies on saccharification and fermentation of dispersed starch-containing raw materials with 8% malt (barley and millet) by weight of dispersed raw materials.

They found that an increase in the activity of malt amiasis by 1.5 - 5% can be achieved by passing an alternating current with a power of 0.013 - 0.015 amperes through the solution. As the current increases, the activity of amylase decreases.

Zabrodsky points out that malted milk, prepared with saccharified mass, improves the saccharification process and the solubility of malt starch.

Table. Extraction of malt amylase.

The study of the duration of saccharification on pure starch solutions showed that changing the duration of saccharification from 5 minutes to 2 hours does not affect the performance of fermented solutions. When saccharifying the boiled mass from grain for 5–45 minutes, a slightly increased content of undissolved starch in mash was observed during rapid saccharification, the amount of unfermented sugars and dextrins was the same. Saccharification of the boiled mass at 55 - 58 ° C for 15 - 120 minutes almost does not cause an increase in the content of fermentable substances in the solution, but with longer saccharification, the concentration of the saccharified mass noticeably increases. So, if after 15 minutes of saccharification the concentration of the saccharified mass was 13.8% (according to the saccharometer), then after 120 minutes it increased to 14.8%.

Thus, when choosing a saccharification mode under production conditions, one should take into account not only the temperature, but also the duration of its exposure, as well as the way the malted milk is processed.

Studies carried out at the Ukrainian Research Institute of Special Use (Raev, Ashkinuzi) showed that when saccharification by a two-stage method, the activity of malt amylolytic enzymes is better preserved, and saccharification in the first stage for 10 minutes and in the second stage for 2 minutes gives a saccharified mass with better indicators than with 10 minutes of saccharification in each stage. From the point of view of increasing the yields of alcohol, two-stage saccharification is more profitable than one-stage.

The studies of Raev, Ashkinuzi, Drazhner and Bazilevich revealed the dependence of the saccharifying and dextrinolitic ability on the method of saccharification.

The same authors found that filtration analysis (determination of filtration rate) can serve as a criterion for assessing the saccharification regime. The table shows the dependence of the filtration rate of the saccharified mass on the duration of saccharification (at a saccharification temperature of 63-64°C).

Table. The rate of filtration of congestion after the 1st stage of saccharification.

The filterability of the saccharified mass is due both to the breakdown of starch into maltose and dextrins, and the accumulation of maltose, which reduces the viscosity of the solution.

The quality of the saccharified mass depends on the mode of digestion adopted.

Zabrodsky and Polozhishnik showed that filtration, spectrophotometric analysis and potentiometric titration can be used for the production characteristics of boiled and saccharified mass.

The table shows the filtration performance of the saccharified mass at a vacuum of 800 mm of water.

Table. Dependence of the filtration of the saccharified mass on the boiling temperature.

Pure starch, devoid of proteins and other colloidal impurities, has a greater ability to filter. Saccharified mass is filtered more slowly from normal corn and even more slowly from defective corn, which can be explained by the formation of colloidal substances with greater hydrophilicity (the ability to absorb and retain water).

According to Zabrodsky, in defective grain at high temperature, along with the dissolution and decomposition of protein compounds, there is a synthesis of water-insoluble humus-like substances.

Klimovsky, Konovalov and Zalesskaya found that during the saccharification of boiled mass, the amount of soluble nitrogen increases due to the action of malt proteolytic enzymes, depending on the accepted temperature regime for boiling grain raw materials.

The largest amount of soluble nitrogen (75% of the total nitrogen of the raw material) is formed at a boiling temperature of 150°C and the smallest (32.8%) - at a temperature of - 100°C. With an increase in the boiling temperature to 120 - 140 ° C, the amount of soluble nitrogen is 40 - 41.9%.

Thus, the native proteins of starchy raw materials are better broken down by malt enzymes than the proteins of heated raw materials.

The hydrolytic splitting of some of the proteins and fats of the grain, the breakdown of carbohydrates, the release of phosphoric acid from organic and inorganic compounds contributes to the formation of substances with acidic properties.

The acidity of the saccharified mass from defective grain is 1.5-2 times higher than the acidity of the saccharified mass from normal grain. The change in acidity depending on the conditions of raw material digestion is graphically shown in the figure.

The color of the saccharified mass can serve as a certain criterion for the processes that occur when the grain is heated. With an increase in the boiling temperature, the mass acquires a straw-yellow and brown color of varying intensity. by color it is possible to a certain extent to judge the quality of the boiled mass. The figure shows a graph showing the dependence of the color of the saccharified mass on the boiling temperature.

For saccharification of starch of grain-potato raw materials, a mixture of barley, millet and oat malts is used, and the sum of millet and oat malts must be at least 30%. It is allowed to use a mixture of two malts: barley and oat or millet. Barley malt can be replaced with rye (or wheat) in whole or in part, and millet malt with chumiza malt. It is forbidden to use malt from one crop in the production of alcohol from grain of the same crop (Smirnov V.A., 1981)....