Nutritional additive for alcohol fermentation medium

FIELD: food industry.

SUBSTANCE: additive is produced from wheat offal fermented with mould fungi. The additive agents are at least one ferment and a mixture of nutritional ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids. The said additive is also used for activation of alcohol fermentation or pre-fermentation intended for preparation of yeast under aerobic conditions.

EFFECT: reduction of the simultaneous saccharification-fermentation stage time in the process of ethanol production, increase of yeast growth and efficiency.

21 cl, 2 dwg, 4 tbl, 4 ex

 

The invention relates to a nutritional Supplement, which creates favorable conditions for the industrial production of ethanol through fermentation, more specifically, the method of simultaneous saccharification and fermentation into alcohol fermentive carbohydrate feedstocks such as starchy raw materials, particularly grain, more specifically, wheat and waste production (grain, bran), if necessary, after saccharification. The invention also relates to a pellet, obtained by carrying out this method, and the method of manufacturing yeast under aerobic conditions.

The known process of ethanol production from starchy materials by enzymatic method including the transition of the starch in the liquid phase with alpha-amylase, which is used for solubilization and hydrolysis of starch and turning it into dextrins. A classic method for the conversion of dextrins into glucose for this stage is stage saccharification using glucoamylase, which is also called amyloglucosidase. Thus, the glucose content at the end of the saccharification is almost 100% of glucose equivalent, corresponding to the amount of starch in the above starchy material. In other words, the entire glucose potential used starchy material is converted into glucose.

Then the glucose is fermented in the tech is in the fermentation stage, during which it is converted into ethyl alcohol under the influence of yeast under anaerobic conditions.

Research on the improvement of this enzymatic method consisted in attempting to merge stages of fermentation and saccharification. In this regard, scientists have tried to carry out the fermentation and saccharification simultaneously during a single stage, called stage saccharification-fermentation", in English "simultaneous saccharification and fermentation" (state owned oof). The glucose content in the medium at the initial stage of this single stage, i.e. after the completion of the transition in the liquid phase, typically less than 3% of glucose equivalent, corresponding to the amount of starch in the above starchy material.

Also conducted experiments with the aim of limiting the duration of stage saccharification so that the glucose content after the completion of this stage was below 100% of glucose equivalent, corresponding to the amount of starch in the above starchy material. In this case, the step of saccharification was defined as "pre-saccharification" or "partial saccharification".

As will be shown in more detail in the description below, the methods for producing ethanol, comprising the step of incomplete saccharification, and not even providing stage saccharification, hereinafter referred to as "methods with incomplete saccharification", predpolagajut the existence of certain limitations. Therefore, the technical solutions adopted for traditional methods of production with a full saccharification before fermentation, a priori can not be used for these methods.

In terms of methods with incomplete saccharification environment in which fermentation occurs and at least part of the process of saccharification, called "environment saccharification-fermentation".

There is a continuing need to increase the yield of ethanol in the methods of ethanol production, more specifically, in case of incomplete saccharification.

The present invention is to meet this need.

According to the invention this problem is solved by using nutritional supplements for the environment saccharification-fermentation conditions, methods of producing ethanol by fermentation of starchy raw materials, which includes, and preferably consists of active natural substances obtained from the fermentation of mold fungi. It can be in liquid or solid form.

As will be shown in more detail in the description below, the presence of such nutritional supplements in the environment saccharification-fermentation is very beneficial to the growth and efficiency of yeast. This resulted in a significant increase in yield in the process of making ethanol from starchy raw materials.

Preference is sustained fashion the method according to the invention includes one and preferably more of the following optional characteristics:

- A nutritional Supplement is a solid or liquid mixture of the above-mentioned active natural substance and at least one useful enzyme. The concept of a useful enzyme includes, more specifically, one or more enzymes with amylase activity, namely, glucoamylases, proteolytic or xylanase activity, or a combination thereof.

- Active natural substance that is present in the nutritional Supplement according to the invention, is in the raw form, i.e. it contains the substrate used during fermentation of the above-mentioned mold fungi, or in the form of extract (extract), i.e. separated from the above-mentioned substrate. If necessary, it is mixed with additional enzymes. Preferably, the substrate is wheat bran.

- Active natural substance selected from the group of substances which includes ergosterol, N-acetylglucosamine, vitamins, in particular vitamin, nucleic acids, amino acids and, preferably, mixtures thereof. Among the amino acids preferably in the presence of one or more, preferably all of the following amino acids: alanine, arginine, asparagine, aspartic acid, poured, leucine, glutamic acid. Among the vitamins celebrated among others, the presence of various b vitamins, for example, B1, B2, B3, B6 and iNOS is the vitamin E.

- Nutritional Supplement for its introduction into the environment saccharification-fermentation shows the following enzymatic activity, which is in particular due to the appropriate selection of the above-mentioned fungi:

- glucoamylase: at least 500 units glucoamylase activity, preferably 750 units glucoamylase activity, more preferably 1,500 glucoamylase activity per gram of dry matter and/or preferably,

- proteolytic: at least 100 units of proteolytic activity, preferably at least 400 units of proteolytic activity per gram of dry matter and/or preferably,

- xylanase: at least 100 units of xylanase activity, preferably at least 400 units of xylanase activity per gram of dry substance.

- A nutritional Supplement derived from the fermentation, preferably, in a solid medium, substrate, preferably, wheat bran with mold fungi, more specifically, selected from among strains of Aspergillus niger, preferably from among the strains of ATSS 201202, ATSS 76060, ATSS 76061, MUCL 28815, MUCL 28816, NRRL 3112 or strains of Aspergillus wholesale, preferably from among the strains of ATSS 22788 and ATSS.

- Nutritional Supplement obtained using the method according to the invention, comprising the following steps:

. Providing wheat bran,

ii. Hydrating, acidification to pH 4 to 5, preferably nitric acid (HNO3) and temperature treatment of the bran so that pasteurized or sterilized with heat treatment should preferably be carried out after moistening,

iii. I received wheat bran strain of Aspergillus niger, selected from among strains of ATSS 201202, ATSS 76060, ATSS 76061, MUCL 28815, MUCL 28816, NRRL 3112 or strain of Aspergillus orizae, selected from among strains of ATSS 22788 and ATSS,

iv. Fermentation of wheat bran in solid state fermenter with periodic stirring at a temperature of from 28°C to 38°C and relative humidity from 45% to 65%, preferably, from 50% to 60% by weight in terms of aeration sufficient to prevent permanent accumulation of carbon dioxide in the thickness of the wheat bran, until the moment when the fermentation will have the following minimum values of enzyme activity:

- glucoamylase: at least 500 units glucoamylase activity, preferably 750 units glucoamylase activity, more preferably 1,500 glucoamylase activity per gram of dry matter and/or preferably,

- proteolytic: at least 100 units of proteolytic activity, preferably, 400 units of proteolytic asset is awns per gram of dry matter,

- silananda: at least 100 units of xylanase activity, preferably, 300 units of xylanase activity per gram of dry substance.

- Nutritional Supplement is the enzyme complex obtained by the method described in the application US 2002 037342.

The invention also relates to a method for producing ethanol from a carbohydrate material, more specifically, of the starchy material, including after the phase transition in the liquid phase stage saccharification and then the fermentation stage, or after the phase transition in the liquid phase and, if necessary, the pre-saccharification step saccharification-fermentation in the environment saccharification-fermentation, the glucose which in the early stage of saccharification-fermentation is not more than 95% of the glucose equivalent, corresponding to the amount of carbohydrate material, more specifically, starch in the above starchy material. The method according to the invention is characterized by the fact that in a fermentation medium or Wednesday saccharification-fermentation enter the nutritional Supplement according to the invention. This introduction can be carried out in a fermentation medium or Wednesday saccharification-fermentation or during the previous stage.

Surprisingly, the presence in a fermentation medium or in any environment of saccharification-fermentation nutrient supplements on izaberete the Oia and more specifically, wheat bran, fermented mold fungi, very beneficial to improve the efficiency of yeast. The latter is transformed into ethanol more glucose with more favorable kinetics.

Preferably the method according to the invention includes one and preferably more of the following optional characteristics:

The glucose content in the early stage of saccharification-fermentation is not more than 3%, preferably 2%, and most preferably not more than 1% of glucose equivalent, corresponding to the amount of starch in the above starchy material. In other words, the method does not include any stage of saccharification or pre-saccharification.

- If a nutritional Supplement is introduced in the raw form, the amount of this additive is higher or equal to 4 kg of dry matter, preferably 14 kg of dry matter and/or less than or equal to 60 kg of dry matter, preferably 34 kg dry matter, more preferably less than 19 kg of dry matter per ton of starch, most preferably about 17 kg of dry matter per ton of starch.

- Nutritional Supplement is prepared by fermentation of a substrate, identical carbohydrate material used in the method of manufacturing ethanol.

- Stage saccharification-fermentation is performed without under the Chi air or oxygen (no aerobic phase in the proper sense).

- Stage saccharification-fermentation includes an initial aerobic phase.

- As agent for ethanol fermentation uses yeast, in particular, yeast of the genus Saccharomyces, more specifically Saccharomyces cerevisiae. More preferably, the yeast worked at concentrations of ethanol above 95 g/l

In this way the above-mentioned nutritional Supplement is a main food source for yeast and/or primary source of ergosterol and/or the main source of nitrogen and/or amino acid and/or phosphorus and/or sulfur and/or vitamins. Preferably, it represented the only source of ergosterol and/or nitrogen and/or amino acid and/or phosphorus and/or sulfur and/or vitamins.

The method according to the invention allows also to obtain a pellet with excellent nutritional value. Thus, the invention also relates to a pellet, obtained by carrying out the method of producing ethanol according to the invention and containing more than 5 g, more preferably 20 g and/or less than 100 g, preferably less than 35 g, most preferably about 30 g of ergosterol per ton of dry matter of grain.

Preferably the above-mentioned grain also contains more than 40%, preferably more than 50% crude protein, calculated on the dry substance. As will be shown in the description below, such a high content of protein was is possible through the use of part of the hemicelluloses and fibers during the pre-fermentation or saccharification-fermentation.

Finally, the invention, in General, refers to the use of a nutritional Supplement according to the invention for promoting alcoholic fermentation in the manufacture of ethanol or pre-fermentation in the production of yeast.

Other characteristics and advantages of the present invention will be elucidated in the description below and in the consideration of the attached drawing, in which figure 1 is a diagram of part of a method of producing ethanol according to the invention.

The methods for the production of ethanol by fermentation of carbohydrate materials, in particular, starchy materials, well known to specialists, they can be adapted and adjusted in accordance with the method according to the invention. So, specialist if necessary able to clarify some points of the description below.

The following relates to the manufacture of ethanol from wheat, but can be applied to any type of starchy material, in particular, to any grain extract. Therefore, the following methods of implementation are only examples, they can be modified, in particular by substitution of technical equivalents, without departing from the scope of the invention.

Method of producing ethanol from wheat according to the invention may, for example, include the following steps:

a) the Step of conditioning the wheat for making milled product p is nichego starch,

b) Phase transition of starch in the liquid phase (liquefaction), in particular, in the presence of alpha-amylase to hydrolyze the starch and turn it into dextrins,

c) optional pre-saccharification, in particular, with the help of enzymes for hydrolysis of dextrins and turning them into formatiruem sugars (glucose, maltose, maltotriose) and non-starchy components and

d) Stage saccharification-fermentation in the environment saccharification-fermentation, containing dextrins and/or the above-mentioned formatiruem sugar and yeast for ethanol production.

These different stages are presented in figure 1.

Step a) consists in making the product of grinding wheat starch, such as flour, by conditioning the aforementioned wheat.

Then wheat flour is mixed with water in the mixer, optional with Barda acid and hydrolytic enzyme so as to form a "thick wort".

Usually thick wort contains from 25 to 35% solids by weight, preferably from 30 to 35%. The solids content was set at this level to limit energy costs while maintaining satisfactory fluidity. For economic reasons the solids content was increased to the highest possible level to limit evaporation costs bards who and subsequent stages of production method. The amount of flour supplied to the mixer is regulated either by a measuring device, or by using the weighing conveyor.

Depending on the hydrolytic enzyme is necessary to adjust the pH by adding an acid solution such as sulfuric acid. Depending on the enzymes in the classic version of bacterial alpha-amylases, more specifically, heat-resistant, if necessary, you can apply a salt of calcium. The consumption of acid is controlled by the pH sensor installed in the pipeline for a mixture of water/bards before thickener. the pH in the classic version can vary from 5 to 6.5, depending on the enzymes used.

After this transition in the liquid phase (phase b)is carried out at a temperature from 80°C to 95°Shustoke wort can be brought to this temperature by direct injection of steam into the reservoir, where the transition occurs in the liquid phase, pipes or steam boiler. In the second case, thick wort for a few seconds is brought to a temperature of from 100°C to 150°C due To the steam supply in the pipeline before rapid cooling to a temperature of from 80 °C to 95 °Caderzone in tanks, where the transition occurs in the liquid phase, can be mixed.

Preferably, the material on the phase transition in the liquid phase has the following options:

Those who temperature: 80°C to 90°C

- pH: 5,5-6,5

- Solids content: 30%-35%

- Time: from 30 minutes to 2 hours.

Step b) leads to the hydrolysis of starch and its transformation into dextrins. When a pre-saccharification (stage C), the liquid wort is cooled in heat exchangers plate or tube type to a temperature of from 50°C. to 60°C.

In some cases, dilution of the wort diluent, such as water or reverse bard or phlegm formed in the distillation installation.

In optional step (C) in the liquid wort is preferably introduced enzyme complex, which has the desired enzymatic activity.

The amount of feed enzymes preferably depends on the amount of wort obtained in the glucose concentration and the residual starch content. The purpose of such adjustment ratio is the preparation of a solution, in which at the end of fermentation will not be starch.

Preferably, the material is pre-saccharification (C) has the following options:

- Temperature: from 50°C to 60°C

- pH: 4-5

- Stay: to obtain the desired concentrations of glucose, usually less than 24 hours.

The contents of the tanks in which the saccharification pre, is subjected to mechanical stirring, which provides a sufficient homogenization of the must is during saccharification, and promotes contact between the enzymes and dextrins subjected to hydrolysis.

The use of enzyme complex obtained from the fermentation of wheat bran mold fungi, preferably allows to reduce the viscosity osaharennogo wort and increase the nitrogen content in the above-mentioned wort.

According to the invention, the saccharification not continues until complete hydrolysis of dextrins and conversion into glucose, and is interrupted at the stage of incomplete hydrolysis. According to the invention, the glucose level at the end of the pre-saccharification below 95%, more preferably below 50%, most preferably below 5%. In this regard, the hydrolysis proceeds at the stage of saccharification-fermentation.

If the method according to the invention does not include a step of partial saccharification or pre-saccharification", the content of glucose in the early stage of saccharification-fermentation d) is not more than not more than 3%, preferably 2%, and most preferably not more than 1% of glucose equivalent, corresponding to the amount of starch in the above starchy material.

At stage d) must combine with yeast in the environment saccharification-fermentation. It is also combined with the nutritional Supplement according to the invention. The order of introduction of yeast and additives does not matter.

The mixture is stirred during the entire process (d).

M is tenderly to use all the yeast, used in ethanol production, in particular a yeast of the genus Saccharomyces. Typically, the yeast is introduced into the environment saccharification-fermentation in aerobic conditions. During this stage, a certain number of divisions of yeast cells. In this case, the yeast converts glucose into ethanol, but rather use glucose for their growth. It is known that to improve the growth of cells should be introduced into the environment saccharification-fermentation nutrient supplements. However, to limit the growth of cells consuming glucose, thereby reducing the yield of ethanol, it is preferable to sow the environment saccharification-fermentation so many yeast to established these yeast ethanol was rapidly reached a concentration in culture medium, which inhibits the growth of yeast cells.

In the first embodiment, the step (d) starts in aerobic conditions during the time required for adequate reproduction of yeast.

Then Wednesday saccharification-fermentation is translated in anaerobic conditions. At this time, the yeast converts glucose into ethanol.

According to the second variant of step d) is performed under anaerobic conditions. The presence of a nutritional Supplement according to the invention eliminates the need for initial aerobic phase.

Preferably, the material at the stage of saccharification-fermentation d) has the following parameters : the s:

- Temperature: 30°C to 35°C

- pH: adjusted by adding acid (e.g. sulfuric acid) at the beginning of step d) to a level of from about 3.5 to 5, preferably from about 3.8 to 5, more preferably from about 4 to 5 and most preferably from about 4 to 4.5.

After adjusting pH at the beginning of stage d) due to the buffer effect of nutritional supplementation on subsequent phases of step (d) adjusting the pH is not provided.

- Inoculate yeast: approximately 106up to 5·108CFU of yeast per ml medium saccharification-fermentation, preferably about 107CFU of yeast per ml medium saccharification-fermentation.

The solids content from 20% to 35%, namely from 20% to 30%.

- Stay: from 20 to 72 hours, namely from 20 to 60 hours. The residence time increases with increasing solids content.

Wednesday saccharification-fermentation according to the invention contains nutritional Supplement according to the invention. This additive can be entered directly into the environment saccharification-fermentation or during the previous stage. Surprisingly, adding on Wednesday saccharification-fermentation of such nutritional supplements, more specifically, wheat bran, fermented mold fungi, very beneficial to improve the efficiency of yeast. The latter is transformed into ethanol greatest number is about glucose with more favorable kinetics. In addition, a nutritional Supplement according to the invention allows to reduce the duration of the aerobic phase in the early stage of saccharification-fermentation or even eliminate it. Preferably, this increases the performance of the process. More preferably, the nutritional Supplement according to the invention allows to reduce cell death compared with the same culture, cultured in the absence of the above-mentioned nutritional supplements. And, finally, its presence has a buffer action, which eliminates the need to adjust pH.

According to the invention preferably Wednesday saccharification-fermentation initially contains 1000 kg of starch, originally introduced in the environment, from 2.5 kg to 35 kg nutritional additives, more specifically, fermented wheat bran, in particular from 8 to 10 kg nutritional additives, more specifically, fermented wheat bran per 1000 kg of starch.

Thus, the presence in the environment saccharification-fermentation fermented wheat bran significantly reduces the time saccharification-fermentation.

Apparently, this nutritional Supplement contributes to the optimal value and the optimal form of nutrients that are great for yeast, in particular, for anaerobic cultures.

Without being bound by theory, the inventors explain this the results as follows. They discovered the presence of a nutrient additive used according to the invention, the amino acids necessary for yeast. These amino acids, apparently, largely contribute to the nitrogen nutrition of the yeast, in particular, is much more effective than simple ammonium salts that were used up to the present time, among other things, reducing the synthesis of by-products formed during the fermentation process, such as glycerin, and really increasing the yield of ethanol.

Increased efficiency of yeast can also be explained by the presence of vitamins in the nutritional Supplement according to the invention.

Finally, the inventors have found that a nutritional Supplement according to the invention contains ergosterol and N-acetylglucosamine, which are important components of the yeast. Their presence in the environment saccharification-fermentation contributes to the growth and success of yeast in total anaerobiosis. Indeed, in these conditions, its synthesis by yeast impossible. Thus, preferably the presence of ergosterol can reduce, or even eliminate aerobic phase at the beginning of the saccharification-fermentation.

However, the tests presented in table 1 below, showed that adding the identified nutrients does not lead to the formation of equally productive environment saccharification-fermentation, and d is the addition of nutritional supplements according to the invention. Thus, in the basis of the received exceptionally high performance lies in the combination of the aggregate components of this additive in proportions resulting from fermentation, mold fungi, and preferably they are filed in the form of wheat bran.

A nutritional Supplement may not be special or optimal enzymatic activity. Therefore, the addition of enzymes.

However, preferably, the nutritional Supplement according to the invention showed enzymatic activity, are useful within the methods of producing ethanol. In particular, it is preferable that a nutritional Supplement showed glucoamylase activity is above 500 units glucoamylase activity per gram of dry matter. More preferably, the nutritional Supplement showed proteolytic activity above 100 units of proteolytic activity per gram of dry matter and/or xylanase activity of at least 100 units of xylanase activity per gram of dry substance.

So, despite the fact that provides for the use of other enzymes, such as purified enzymes or complexes of purified enzymes, preferably according to the invention to use fermented nutrient additive under conditions that allow her to Express the above-mentioned enzymatic aguosti.v this case, this nutritional Supplement as enzyme complex must be typed preferably in step (d), and/or optionally, in stage C).

Preferably, the nutritional Supplement is a fermented wheat bran, which were prepared or can be obtained using the following method according to the invention:

i) providing wheat bran,

ii) hydrating and temperature treatment of the bran so that pasteurized or sterilized with heat treatment should preferably be carried out after moistening,

iii) sowing received wheat bran strain of Aspergillus niger, selected from among strains of ATSS 201202, ATSS 76060, ATSS 76061, MUCL 28815, MUCL 28816, NRRL 3112, preferably from among the strains of ATSS 76061 and NRRL 3112, more preferably a strain of ATSS 76061, or from a number of strains of Aspergillus orizae ADS 22788 and ATSS 42149.

iv) fermentation of wheat bran in solid state, preferably in the form of a layer thickness of more than 10 cm in the fermenter with periodic stirring at a temperature of from 28°C to 38°C and relative humidity from 45% to 65%, preferably, from 50% to 60% by weight in terms of aeration sufficient to prevent permanent accumulation of carbon dioxide in the thickness of the wheat bran, until the moment when the fermentation will have the following minimum values of the enzymatic activity is STI:

- glucoamylase: at least 500 units glucoamylase activity, preferably 750 units glucoamylase activity, more preferably 1,500 glucoamylase activity per gram of dry substance, and preferably,

- proteolytic: at least 100 units of proteolytic activity, preferably, 400 units of proteolytic activity per gram of dry substance, and more preferably

- silananda: at least 100 units of xylanase activity, preferably, 300 units of xylanase activity per gram of dry substance.

In phase i) wheat bran preferably chosen so that the fraction of particles smaller than 1 mm was at least 40% by weight.

In phase ii) wheat bran must be wetted and subjected to thermal processing for the purpose of pasteurization or sterilization. Preferably, this heat treatment was not preceded by hydration, as in the case when conducting a thermal treatment of the bran before hydration, there were not enough satisfactory fermentation.

Thermal processing may be heated, for example, in an autoclave. The autoclave for 20 minutes at a temperature of from 120 to 121°C gave very satisfactory results. Well me is it hard conditions of pasteurization in a drying Cabinet at 105°C for 15 minutes You can also conduct thermal processing bran, giving bran pairs, this allows you to moisturize bran.

Preferably carry out the pH adjustment, preferably nitric acid in the wetting time in the range from 4 to 5.5 to strengthen posterizeimage effect of thermal processing and launch the desired fermentation. Particularly preferably nitric acid, because it is also used as a nitrogen source for fungi.

In addition to the sterilization temperature processing also contributes to the gelatinization of the starch contained in wheat bran and, consequently, the availability of this substrate for Aspergillus niger and Aspergillus orizae, which increases the efficiency of fermentation.

Important procedure wetting bran, as the water content affects the results of the fermentation. Hydration is conducted so that the water content in the bran at the beginning of the fermentation stage iv) was in the range of 50-60%, preferably 50-55% of the total mass of bran and water.

At stage iii) sowing wheat bran can be performed using any suitable inoculum. The expert knows different methods of preparation of suitable seed material on the basis of the selected strain. Preferably, the seeding rate is at least 107spores/gram of the outcome of the s dry substances.

At stage iv) fermentation can be carried out in any suitable fermenter. Examples of fermenters that can be used, as described in article Aduana and colleagues (A.Durand et Coll.), published in Agro-Food-Industry Hi-Tech (may-June 1997, p.39-42).

Fermentation should be carried out until the moment when glucoamylase activity will be at least 500 units glucoamylase activity, preferably at least 750 units glucoamylase activity, more preferably at least 1500 units of glucoamylase activity per gram of dry matter of bran, that is usually during the period from 1 to 3 days, preferably from 30 to 60 hours. Fermentation, lasting less than 1 day too incomplete. After 3 days fermentation is stopped or almost ends, and thus its continuation becomes uneconomic.

The temperature is maintained in the range from 28°C to 38°C, preferably from 32°C to 36°C, which corresponds to the known optimal area of activity of the used strains. Preferably, the temperature during the first hours of fermentation set in the range from 34°C to 38°C to facilitate spore germination, then the rest of the fermentation stage was lowered to 28°C-32°C in order to facilitate the adjustment of the temperature of the environment.

the contents of moisture in wheat bran is usually from 50% to 60%. However, humidity can deviate within ±5% of the range of 50-60% for a relatively short time between two consecutive adjustments of humidity or at the end of fermentation. In any case, it is sufficient that the moisture level did not fall below 45%. Under cultivation the moisture level of culture medium tends to decrease due to evaporation by heat, which is released during the growth of fungi, as the environment is a poor conductor of heat. Therefore, during fermentation it is necessary to maintain a certain moisture content, for example, periodically adding water to the environment to compensate for the loss of water environment. The quality of the water also plays a very prominent role. You can use the tap water is of good quality or distilled water.

The PH of the fermentation medium is typically not adjusted. As explained above, preferably the level is adjusted initially in the range from 4 to 5.

If the first pH is about 6.0 and about 6.4, under cultivation, the pH decreases to 3.8 to 4.2, and then by the end of rises again. This second increase, as a rule, is correlated with the phase of sporulation of the fungus. Monitoring of the dynamics of pH is a means of determining the state of the culture.

The fermenter should tank, preferably the village is constantly to to ensure the supply of oxygen necessary for fermentation, and to prevent an excessive accumulation of carbon dioxide generated during fermentation. In addition, aeration is involved in regulation of temperature and humidity culture medium. The air should preferably be saturated to limit the tendency of the drying environment. It is difficult to give quantitative guidance on the stream aeration, because it involves many variables, in particular, the size and geometry of the fermenter, the number of downloaded bran and the like. However, a specialist with the help of simple routine experiments will be able to determine the appropriate flow aeration in each case; usually a sufficient flow of air from the calculation of 1-2 m3per hour per kg of dry substances, excessive pressure should preferably be from 0.5 to 1 bar.

The party loaded in the enzyme bran during fermentation must be periodically stirred using a stirring rods, knives or blades, or using screws to prevent the formation of an impenetrable mass and to ensure the most uniform aeration of the total weight of the bran. Used strains surprisingly resistant to mixing. However, you should avoid overly intensive mixing.

Fermented wheat bran used in the method according to the invention, if desired, can be dried or frozen for preservation or refrigerated and used without additional processing.

Drying is preferably carried out at moderate temperatures, so as not to disrupt enzymatic activity. The appropriate way is to heat in a drying Cabinet at 40°C. on an industrial scale it is preferable to vent the dry air at a temperature from 35°C to 40°C depending on the mold fungus. In turn, freezing at a low temperature, for example, at -20°C can be subjected to wet the product.

US 2002037342 discloses an enzyme complex with glucoamylase, proteolytic and xylanase activity obtained during the fermentation of wheat bran with strains of Aspergillus. This complex can be used as a nutritional Supplement according to the invention.

As a rule, in the framework of the method including the step of saccharification, at least in the part thereof which is carried out simultaneously with the fermentation, it is impossible to foresee the action of the enzyme or mnogovershinnoe composition, which is known under way with separate stages of saccharification and fermentation. In particular, this is confirmed by the fact that when adding the composition, for example, compositions according to US 2002037342, implies the inclusion of substances such as hemicellu is for, capable of increasing the viscosity during fermentation.

This document describes the use of this composition to improve conditions at the stage of saccharification. It in no way means that the composition may be of interest for the method, not including the stage of saccharification or only one pre-saccharification.

Indeed, the optimal conditions for saccharification and fermentation vary greatly. In particular, the saccharification often conducted at a temperature of about 60°C, i.e. at a temperature which is not suitable for yeast, usually used during fermentation. Mutually, as illustrated by table 7 of document US 2002037342, the enzyme composition according to US 2002037342 are optimal enzymatic activity at a temperature saccharification, but, as expected, show a reduced enzymatic activity in the case of the simultaneous saccharification-fermentation.

In addition, it is known that the enzyme composition according to US 2002037342 reduce the viscosity of the medium saccharification at a temperature saccharification (see table 8), but with probability can expect that this reduction in viscosity will not be played at a temperature of fermentation, at least, will be played to the extent that will make acceptable medium viscosity saccharification enzyme is AI.

In this regard, the expert will not attempt to use the composition according to US 2002037342 in the method of producing ethanol, not including the stage of saccharification or only one pre-saccharification. It is even less reasonable in light of the fact that the exception saccharification or limit its duration can sometimes lead to the formation environment saccharification-fermentation incorrigible contaminated, which can be improved only by adding large quantities of bacteriostatic drugs. Indeed, the temperature saccharification provides protection against bacterial contamination.

The invention also relates to the use of wheat bran obtained in the above way, or way that is consistent with wheat bran as described in US 2002037342, to create favorable conditions for alcoholic fermentation in the environment saccharification-fermentation. Preferably at the beginning of stage d) in the environment saccharification-fermentation must be more than 4 kg of such bran per 1000 kg of starch.

Mainly in the preferred method of the invention, the nutritional Supplement is mnogovalentnym complex, obtained by fermentation of wheat bran mold fungi and:

- is a means of increasing the value of the bran obtained from step a) preparation of p is technoi flour,

makes a number of useful enzymes, thus, simplifying the way, and

- is an excellent nutritional Supplement for yeast. However, to ensure this last effect mass fraction mnogovershinnoe complex should be much higher than in the preceding reception.

Modern studies have the tendency to manufacture more and more concentrated mnogovalentnykh complexes in order to limit the mass fraction of the injected substances. This leads to the simplification methods.

In contrast to the above mentioned trends inventors, in contrast, revealed that the introduction of more than 4 kg of dry matter, preferably more than 14 kg of dry matter, more preferably about 19 kg of dry matter fermented wheat bran per ton of initial starch can improve the overall productivity of the method of manufacture of ethanol.

Output after step d) fermented wort or wine, obtained by saccharification-fermentation, after passing through the heat exchanger is fed to the distillation column.

Bard extending from the base of the column is sent to the workshop of the Department of grains for purification by classical methods, centrifugation, with the aim of separating soluble from insoluble substances.

Raw grain, the resulting phase separation, sistotrema from 35% dry matter, the clarified liquid grains contain from about 7% to 10% of dry substance.

The clarified liquid bard concentrated by evaporation in vacuo to obtain a syrup or concentrated stillage with a solids content of about 35%.

The resulting syrup can be mixed with raw grains (solid phase). The mixture is then dried and receive about 350 kg of grains, dried thus, per kg of wheat, and the solids content in the grain is about 90%.

The pellet obtained after drying, has excellent nutritional properties, in particular, for cattle, it is also an object of the invention. Indeed, 350 kg of grains obtained according to the invention contain from 2 to 35 g of ergosterol, preferably about 10, so as ergosterol is a precursor of vitamin D2, he, therefore, is of interest for health, as well as interest for yeast nutrition, particularly in anaerobic conditions.

In addition, in the case where the nutritional Supplement is a wheat bran, fermented according to the above method according to the invention, the resulting pellet contains an excess of protein.

Alcohol vapor from the column is condensed in the heat exchangers. Azeotropic mixture captured in the upper part of the column, dried using classical methods, nab is emer, using a molecular sieve.

The method according to the invention allows to obtain from about 375 to 390 liters of ethanol from the original 1000 kg of wheat. In other words, an amazing way to 91% of the stoichiometric yield of ethanol from wheat, containing about 60% of starch, in the conversion of glucose into ethanol by fermentation.

Without being bound by theory, the inventors explain these results by replacing the introduction of exogenous nitrogen, for example, in the form of sulphate of ammonia, free amino nitrogen. This replacement preferably reduces the synthesis of side-enzyme products, in particular, glycerol, and, consequently, increases the product yield.

The invention relates also to a method of manufacturing yeast under aerobic conditions or reproduction of yeast", which more specifically can be used for pre-fermentation with the aim of preparing yeast, which is then placed in a fermentation medium or Wednesday saccharification-fermentation method the alcoholic fermentation of glucose to ethanol.

In this way the pre-fermentation usually aims to increase the concentration of yeast in the environment of pre-fermentation level of about 106-107CFU per ml at least about 108CFU per ml of the medium prior to fermentation, preferably at least approximately 5·108Cohen ml of medium prior to fermentation.

Pre-fermentation is carried out in fermenters for pre-fermentation, where the temperature is strictly controlled and regulated, for example, by means of system of cooling plates to the outside or inside of the tanks prior to fermentation, in which circulates the coolant. Indeed, any reproduction of microorganisms leads to an increase in temperature, which may be an inhibitor of reproduction of yeast. Classically the temperature in the fermenter for a pre-fermentation supported in the range from 30°C to 35°C.

For the propagation of yeast is necessary the introduction of oxygen, for example in the form of compressed air.

It is known that to facilitate the development of yeast in a nutrient medium should be added:

- nitrogen, applied in various forms, such as urea, ammonia, ammonium salt,

phosphorus, which is included in various forms, such as phosphoric acid, phosphates,

- sulfur, payable in various forms, such as sulfuric acid, sulfates,

the main minerals.

By the previous method in the nutrient medium also add the wort leaving after phase transition in the liquid phase, or pre-saccharification saccharification. It must also contributes formatiruem sugar, but reduce the overall efficiency of alcoholic fermentation. T is thus, there is a need in the method of manufacturing yeast under aerobic conditions, which can be used at the stage of preliminary fermentation for the preparation of yeast, which is then placed in a fermentation medium or Wednesday saccharification-fermentation method the alcoholic fermentation of glucose to ethanol, which would limit this reduction efficiency.

According to the invention this problem is solved by adding in the fermenters for pre-fermentation, at least part of the clarified stillage, more specifically,

bards obtained at the output of phase separation, and/or concentrated stillage obtained in the process of making ethanol from carbohydrate raw materials, more specifically starchy raw materials through fermentation and/or method according to the invention.

Bard can be obtained through methods of producing ethanol, comprising the step of saccharification-fermentation, and the way in which the stages of the saccharification and fermentation are completely separate.

Preferably the bard is obtained in the framework of the method of producing ethanol according to the invention, in which was introduced a nutritional Supplement according to the invention.

Thus, the invention also relates to such methods of producing ethanol, as described above, in which for ethanol fermentation uses yeast obtained in this way OPI is ANO above, and uses the bard, obtained by carrying out the aforementioned method of manufacturing ethanol.

A special advantage is the use of bards, obtained by carrying out the method of producing ethanol according to the invention, in which was introduced a nutritional Supplement according to the invention for the production of yeast in the fermenter prior to fermentation, as it allows to separate the ethanol production by yeast, on the one hand, and growth of yeast cells, on the other hand. Indeed, bard, obtained by carrying out the method of producing ethanol according to the invention is a culture medium that allows you to grow the yeast to a level comparable with increased seeding rate environment fermentation or saccharification-fermentation, resulting in the use of yeast formatiruem Sugars present in the fermentation medium or saccharification-fermentation, mainly for ethanol production, and not for the growth of yeast cells, as described above. So, formatiruem sugar from the source of carbohydrate substrate is mainly used for ethanol production and cell growth is mainly achieved through nonfermentable Sugars. Thus, the overall efficiency of ethanol production from a source of carbohydrate substrate is higher in cf the ranking methods, in which formatiruem zaharopoulos yeast simultaneously for ethanol production and cell growth.

A special variant of the method described in figure 2.

Preferably the nutrient mixture prior to fermentation contains grains and released from the intermediate products obtained in the process of making ethanol from starchy raw materials through fermentation, more specifically, the bard is not mixed with the wort obtained during the intermediate stages of a method of manufacturing ethanol them starchy raw materials through fermentation.

According to another preferred variant of the nutrient medium for the pre-fermentation consists of or includes the liquid mixture bards and wort, and, if necessary, water.

According to another preferred variant of the nutrient medium for the pre-fermentation consists of or includes a mixture of bards and nutritional supplements according to the invention, more specifically, fermented wheat bran and, if necessary, water.

Finally, you can also prepare a nutrient medium composed of or comprising the above-mentioned elements.

Nutrient medium for the pre-fermentation according to the invention preferably contains such a quantity of dry substances that mixing and/or aeration of the environment one is camping sufficient to maintain optimal production of ethanol, for example, from 15% to 20% of dry substance.

For the method of preparation of yeast according to the invention nutritional Supplement according to the invention can be fermented wheat bran. Bard, obtained from the fermentation medium or saccharification-fermentation is a composition and, more specifically, a certain amount of Sugars that can be used by the yeast, and this composition is particularly favorable for the development of yeast in the fermenter prior to fermentation. Wednesday pre-fermentation can be supplemented with exogenous nutrients, such as glycerol or solutions of glycerol, and the hydrolysates obtained from raw grain. This allows it is preferable to conserve glucose wort according to the invention and, thus, to limit the above-mentioned reduction in the efficiency.

Preferably, at least a portion of the stillage is recycled through its incorporation into the nutrient medium prior to fermentation. It is also preferable to reuse the grains after concentration to limit the dilution of the nutrient medium in the fermenter prior to fermentation.

Preferably the bard, which is present in the nutrient medium prior to fermentation, completely replacing the wort leaving after phase transition in the liquid phase, saccharification or prefix is inogo saccharification. But such a replacement may be only partial.

Adding a nutritional Supplement according to the invention, more specifically, fermented wheat bran also helps to improve pre-fermentation, in particular, by enriching the nutrient medium:

- nitrogen applied in the form of amino acids,

- phosphorus

gray,

- vitamins and essential minerals

- proteins.

This addition can be performed directly in a nutrient medium prior to fermentation, or after mixing the wort leaving after phase transition in the liquid phase; this mixture, after dilution if necessary, is added to the fermenter prior to fermentation.

The use of yeast pre-fermented with Barda obtained during the implementation of the method of producing ethanol according to the invention preferably also allows you to significantly enrich proteins dried pellet obtained at the end of the method for producing ethanol according to the invention. Thus, the aforementioned grains may contain more than 40% protein, preferably at least 50% protein in the dry matter equivalent.

Different enzymatic activity referred to in the description and in the claims, was measured using the following methods:

Glucoamylase activity.

Under the action of the preparation is that of glucoamylase (HA) on the solution of the soluble starch is the release of reducing Sugars. When heated to 100°C. in the presence of 3,5-dinitrosalicylic acid (BPS), these compositions become brown, which is determined by using a spectrophotometer (company Kontron Instruments, Milan, Italy) at 540 nm.

The reaction medium contains:

the starch solution: 1.5% in the case of Aspergillus niger and 2% in the case of Aspergillus orizae 1000 ál

- 0.1 M nitrate buffer with pH 4.5 900 ál

The enzyme solution 100 ál

The reaction proceeds for 20 min at 60°C in the case of Aspergillus niger and within 5 minutes at 50°C in the case of Aspergillus orizae. Sampling of the reaction medium in a volume of 100 ál is held every 4 minutes in the case of Aspergillus niger and every minute in the case of Aspergillus orizae, the sample is mixed with 500 ál of BPS and 400 μl of nitrate buffer with pH 4.5. The mixture is then heated for 5 minutes at 100°C, cooled rapidly and perform quantitative analysis at 540 nm relative to the control sample: a mixture of 500 μl of BPS and 500 μl of citrate buffer pH 4.5.

Conditions for quantitative analysis were established after studying the effect of temperature and pH on the activity of drugs HA. As the substrate enzymatic hydrolysis was used soluble starch Merck (Darmstadt, Germany). The BPS solution was prepared according to the procedure proposed by P. Bernfeld in the journal Methods in Enzymology (Bernfeld, Methods in enzymology, 1, 149-158 (1955)), as follows:

- Dissolve previously:

- 10 the 3,5-dinitrosalicylic acid

- 200 ml of 2M sodium hydroxide, 200 ml of distilled water

- Then add:

- 300 g double potassium-sodium salt of tartaric acid

After complete dissolution pour the resulting solution with distilled water to 1 L.

After preparing this reagent should be protected from exposure to light. Calibration curves were drawn for glucose as a standard product for the quantitative assessment glucoamylase activity or to monitor reactions of transition in the liquid phase - saccharification and xylose to determine xylanase activity.

One glucoamylase activity (EGA) corresponds to the amount of enzyme required to release one micromole reducing ends per minute under the conditions dosed supply of glucose as the standard. Glucoamylase activity, calculated using the following formula, equals the original amount of dry substances (ICB):

A=P ×VfermVenz×Mferm

where

And with the activity of HA, exp is defined in EGA/g ICB (μmol/min·g ICB),

- R corresponds to a rate of release of glucose equivalents in µmol/min

- Venzrepresents the metered volume injected solution of enzyme per ml

- Vferm- this is the total amount of distilled water used for the extraction of a solution of enzymes, in ml,

- Mfermis the mass, expressed in g ICB, is the initial dry mass, from which was extracted the solution of enzymes.

Proteasa activity.

This quantitative analysis was developed for azocasein in accordance with the method of Beinoni (Beinon), described in the publication "Proteins Purification methods - a practical Approach", Harris, E.L.V. and Angal, S (compilers), IRL-Press, Oxford University Press, 1-66 (1989). Decomposition of this substrate by proteases released nitrogenous groups which absorb light in the ultraviolet part of the spectrum at 340 nm. The dynamics of the absorption coefficient at the time of hydrolysis of the protein indicates the intensity of the response.

The reaction medium contains:

the solution azocasein 1%,pH 5.0 OO ál

The enzyme solution 200 ál

Azocasein (Sigma (Sigma), St. Louis, USA) dissolved in acetate buffer with 0.1 mol/l, pH 5.0. Protease activity quantitatively assessed at this level of pH because azocasein insoluble in acetate buffer at lower pH value. The enzymatic reaction is carried out at 60°Sombor about who runs every 5 minutes within 20 minutes; to stop the reaction, samples are mixed with 5% trichloroacetic acid (THUK).

One unit by activity (EEA) corresponds to the amount of enzyme required to increase Azo nm; generated when the release azotistykh groups by 0.01 units per minute under the conditions described above. This activity is calculated using the following formula, equals the original amount of dry substances (EEA/g ISV) or glucoamylase activity (EEA/EGA):

A=P ×VfermVenz×Mferm

where

And corresponds by activity, expressed in EEA/g ICB,

- P corresponds to a rate of release of nitrogen groups, expressed in the increase And340nm 0.01 units / min,

- Venzrepresents the metered volume injected solution of enzyme per ml

- Vferm- this is the total amount of distilled water used for the extraction of a solution of enzymes, in ml,

- Mfermis the mass, expressed in g ICB, is the initial mass of dry substances,from which was extracted the solution of enzymes.

Silananda activity

To detect this enzyme activity drugs HECTARES affect the solution of soluble xylan and measuring the amount of released reducing sugars by the method of using BPS.

The reaction medium contains:

the solution of xylan 2%, pH 4.5 1900 ál

The enzyme solution 100 ál

Prepare a solution of larch xylan (Sigma, 1%) in 0.1 M citrate buffer pH 4.5. The reaction proceeds at 60°C in the case of Aspergillus niger and at 50°C in the case of Aspergillus orizae. Sampling of the reaction medium in a volume of 200 μl is held every 2 minutes for 10 minutes, the sample is mixed with 500 ál of BPS and 300 μl of nitrate buffer with pH 4.5. The mixture is then heated for 5 minutes at 100°C, cooled rapidly and perform quantitative analysis at 540 to them relative to the control sample: a mixture of 500 μl of BPS and 500 μl of citrate buffer pH 4.5.

One unit of xylanase activity (ESA) corresponds to the amount of enzyme required to release one micromole reducing ends per minute. This activity will be calculated initial amount of dry matter (ESA/g ISV) or glucoamylase activity (ESA/EGA). To calculate this activity we took the formula derived for the calculation of the HA, in which:

- And - match xylanase activity, expressed in ESA/g ICB (µmol/min t ICB),

- P - corresponds to the MSE of the spine of the release of xylose equivalents in µmol/ min,

- Other components of the formula have not been modified.

The following non-limiting experiments are given to illustrate the invention, in particular the advantages that is provided through the use of fermented wheat bran according to the invention in the case of the implementation of the method of simultaneous saccharification-fermentation.

Example 1: the additional Effect of nitrogen application

Conducted a comparative study of the amount of ethanol produced during saccharification-fermentation, with the introduction of a commercial enzyme preparation (Spirizyme® Fuel), or the same enzyme preparation with unlimited add nitrogen, or nutritional Supplement according to the invention.

In these experiments, the researchers prepared a 750 g of wort from wheat flour with a content of dry substance 32% in the mixed reactor with a capacity of 1 L. After 2 hours of transition in the liquid phase at 85°C and pH 5.5 wort is poured into 3 erlenmeyers flask 0.5 l, each of which was placed 200 g of wort and created the same conditions for glucoamylase activity as follows:

The flask 1: Spirizyme® Fuel (solution-treated glucoamylase, Novozymes);

The flask 2: Spirizyme® Fuel and nitrogen additive, namely secondary acid phosphate and acidic ammonium sulfate: 3 g/l of each substance;

The flask 3: wheat bran fermented with the strain of Aspergillus niger was ATSS 76061 by the way, opican the mu US 2002037342.

In all three flasks initial pH value was adjusted to 4.5, a temperature of 32°C.

The introduction of nitrogen into the flask 2 resulted in alkalinization of pH to a level of 6.2, which required additional add concentrated hydrochloric acid to reduce the initial pH of the wort to 4.5 for carrying out saccharification-fermentation. Flasks with wort were seeded with yeast at the rate of 107CFU per ml of wort. The examples have used the yeast Saccharomyces cerevisiae.

After complete enzymatic hydrolysis of starch maximum theoretically possible content of glucose in the wort is 303 g/l

As shown in table 1, the maximum yield of ethanol was obtained by adding fermented wheat bran for 72 hours. The use of Spirizyme® Fuel, even with nitrogen additive allows to obtain a maximum of 59% ethanol for 96 hours. In the absence of additional application of nitrogen ethanol production is very small, and we noted increased glucose content.

Table 1
Production. ethanol/finish. volume of ethanol with the enzyme. bran (%)Production. glucose/finish. quantity of glucose with the enzyme. bran (%)
Will continue. The state owned oof (h)SpirizymeSpirizyme + NThe enzyme. branSpirizymeSpirizyme + NThe enzyme. bran
00,00,00,00,016,116,1
10,00,00,018,029,251,3
40,80,61,171,856,8119,5
82,71,26,9143,6to 130.1144,9
114,64,2of 17.5197,5170,8119,9
6,212,130,9269,3110,289,8
208,632,051,0359,166,555,1
249,937,057,1430,93,438,6
30the 13.445,073,6538,62,850,0
36the 13.448,281,3646,47,674,2
4816,453,594,7861,810,671,6
7218,4 57,399,31292,7133,981,8
9616,959,0100,01723,6239,0100,0

Thus, the use of wheat bran, fermented mold fungi, the production of ethanol from wheat provides a significant advantage, even when the temperature in the fermenter during simultaneous saccharification-fermentation. Comparison with Spirizyme Fuel indicates that the unlimited addition of nitrogen only partially explains the increase in the efficiency of the process.

These experiments demonstrate the adequacy of the use of fermented wheat bran for the production of ethanol by the method of simultaneous saccharification-fermentation.

Example 2: the Buffer effect of a nutritional Supplement according to the invention

This example illustrates the advantages of using nutritional supplements of the invention to create favorable conditions for saccharification and alcohol fermentation. More specifically, this example applies to buffer the effect of a nutritional Supplement according to the invention on Wednesday saccharification-fermentation.

In these experiments, the researchers prepared a 750 g with the SLA from wheat flour with a content of dry substance 32% in the reactor with a capacity of 1 l with devices for mixing. After 1 hour 30 minutes transition in the liquid phase at 85°C and pH 5.5 in terms of stirring the wort is poured into 2 erlenmeyers flask of 250 ml, each of which was placed 100 g of liquid wort solids content of 32% and 40 g of sterile water to obtain a wort with a solids content of 23%. Then in each flask with wort added:

- Flask of 1: 0.5 g of wheat bran fermented with the strain Aspergillus niger ATCC 76061 according to the method described in US 2002037342;

The flask 2: 16,7 ál Spirizyme® Fuel (solution-treated glucoamylase, Novozymes).

In both flasks initial pH value of the mash was adjusted to level 4. The wort was seeded with yeast Ethanol Red® (Lesaffre) in amounts of 5-10 CFU per ml of wort.

In flask 2 was added exogenous nitrogen in form of ammonia at the rate of 1 g nitrogen per kg of wheat.

Throughout the experience the mash was maintained at a temperature of 30° under the conditions of shaking at 100 rpm

The desired concentration of ethanol is 87 g of ethanol per liter of wort (11% by volume), which corresponds to a degree of conversion of starch to ethanol 81%.

Table 2 shows the dynamics of the concentration of ethanol in the wort and the pH of the wort depending on the duration of the saccharification-fermentation.

Like table 1, table 2 illustrates that the use of a nutritional Supplement according to the invention in the environment of the saccharification-fermentation allows you to get more height is such that the concentration of ethanol in a shorter time, than using traditional enzymes, despite the introduction of exogenous nitrogen.

In addition, table 2 shows that the introduction of ammonia into the flask 2 leads to lower pH to the level of about 2.8.

For best performance on ethanol, it is important that the pH of the wort remained at the level from 3.5 to 5. Indeed, during the process of saccharification-fermentation at about 30°C. excessively high pH, more specifically, at pH above 5, increases the risk of contamination of the wort. Too low pH level, more specifically, below the 3.5 is a decrease in the efficiency of alcoholic fermentation.

Use of the enzyme preparation, which added exogenous source of nitrogen in the environment saccharification-fermentation requires the device to adjust the pH, which helps to maintain the pH in the range from 3.5 to 4.5.

A nutritional Supplement according to the invention has a buffering effect on the pH of the wort during the saccharification-fermentation and, therefore, preferably eliminates the device regulating the pH.

Table 2
The ethanol concentration (g/l)PH
Will continue. OO is (h) The enzyme. branSpirizyme + NH3The enzyme. branSpirizyme + NH3
00044
1956,6to 33.83,72,9
2872,748
438263,9the 3.82,7
4782,671
678877,9the 3.82,8

Example 3: Biostimulation

This example reveals the biostimulating effect of a nutritional Supplement according to the invention. The experiments in this example illustrate that the addition of nitrogen and the effect of stabilizing the pH of the wort at around 4 only justiceoriented improving the efficiency of yeast in the production of ethanol in their introduction to the environment saccharification-fermentation.

In these experiments, the researchers prepared a mash of wheat flour with a solids content of 32%.. After 1 hour 30 minutes liquefaction at 85°C and pH 5.5 in the conditions of stirring at 250 rpm, the wort is poured into 2 bioreactor with a volume of 4 liters each reactor was 1.7 kg wort solids content 29,8%. The wort obtained in each bioreactor was prepared by mixing the wort with a solids content of 32%, which was formed after the transition into the liquid phase, and bards with a solids content of 27%.

For cultivation wort was used bard, resulting in the implementation of the method of alcoholic fermentation of wheat.

1.7 kg wort solids content 29.8 per cent were made to the following:

- Bioreactor 1: 199 ál of Spirizyme® Fuel (solution-treated glucoamylase, Novozymes) and 59 μl Viscozym Wheat® (Novozymes) (the enzyme that reduces the viscosity);)

- Bioreactor 2: 5.7 g wheat bran fermented with the strain Aspergillus niger ATCC 76061 according to the method described in US 2002037342;

In both bioreactors initial pH value of the mash was adjusted to 4.1, temperature at 30°, then the juice was subjected to shaking at 100 rpm

The wort was seeded with yeast at a rate of 5·106CFU per ml of wort (Ethanol Red®, Lesaffre).

Exogenous nitrogen was added to the bioreactor 1 portions in the form of ammonia so that its content was 1 g nitrogen is per kg of wheat.

Bard, obtained from a mash of fermented grains, contains a number of fibers. When mixed with the wort is achieved by buffering effect on the pH of the wort.

The required concentration of ethanol is 87 g of ethanol per liter of wort, which corresponds to a degree of conversion of starch to ethanol 81%.

Table 3 shows the dynamics of the concentration of ethanol in the wort and the pH of the wort depending on the duration of the saccharification-fermentation.

Portions of the introduction of exogenous nitrogen, and the use of bards allows to stabilize the pH in the bioreactor 1 to about 4.

As can be seen from table 3, the desired concentration of ethanol is almost achieved for 28 hours in the bioreactor with the nutritional Supplement according to the invention, while achieving equivalent concentration in the bioreactor with enzyme preparation required 44 hours. In addition, after 68 hours, the ethanol concentration in the bioreactor with the nutritional Supplement according to the invention is higher than in the bioreactor with the enzyme preparation.

Table 3 illustrates the significant advantage of using a nutritional Supplement according to the invention in comparison with classical enzyme preparations. The results of these experiments indicate that the additional application of nitrogen and pH control only partially explain the effectiveness of a nutritional Supplement according to the invention.

p> These experiments reveal the effect of biostimulation of yeast associated with the composition of nutrient additives according to the invention.

Table 3
The ethanol concentration (g/l)pH
Will continue. The state owned oof (h)The enzyme. branSpirizyme + NH3+ VWThe enzyme. branSpirizyme + NH3+ VW
0004,14,1
86,8<444,1
206935,5a 3.93,75
2473,548,84the 3.8
2882,558,54 a 3.9
4485,682,64,1the 3.8
4893,688,94,13,85
68to 97.1for 91.34,24

Example 4: Aerobic fermentation stillage

The inventors have conducted a comparative study of the dynamics of the concentration of yeast in the environment prior to fermentation, depending on the content of dry substances in the environment.

These experiments were carried out on 100 g environment prior to fermentation, placed in flasks with baffles volume of 250 ml PE fermentation was kept in a thermostat at 30 °C, shaken at 130 rpm and seeded with yeast at the rate 2.5·107per ml of the medium (Ethanol Red®). The initial pH of the media ranged from 4.2 to 44.

Each Wednesday prior to fermentation had the following composition in mass percent:

The flask 1: 1,2% vinasse solids content of 28% and about 98.8% of the water;

The flask 2: 10% vinasse solids content of 28% and about 90% water;

The flask 3: 20% vinasse solids content of 28% and about 80% water;/p>

The flask is 4: 40% vinasse solids content of 28% and about 60% water;

The flask 5: control environment (glucose and yeast extract). The flask was shaken for enrichment of the environment with oxygen and create favorable conditions for the propagation of yeast.

The inventors determined the change in the concentration of yeast in each environment prior to fermentation.

The medium composition and dynamics of yeast concentration are summarized in table 4 below:

Table 4
Number (yeast/ml)
The duration of culture (h)1.2% of bards10% bards20% bards40% of bardsControl
02,50·1072,50·1072,50·1072,50·1072,50·107
53,30·1074,90·1071,54·1084,15·1084,90·107/td>
73,00·1072,50·1083,10·1084,50·1086,00·107
243,30·1071,90·1073,50·1085,50·1082,00·108

As can be seen from table 4, the environment prior to fermentation, consisting of 40 wt.% from Barda with a solids content of 28 wt.%, therefore, the medium containing 11.2 wt.% dry matter, allows for 5 hours at 16.6 times to increase the amount of yeast. For comparison, the ratio of increase over the same period in the control environment amounted to 2.

After 24 hours of cultivation in aerobic conditions, the concentration of yeast in the environment, 40 wt.% consisting of bards, more than 2.5 times higher than the concentration of yeast in the control environment.

Without being bound by theory, the inventors explain these results by the presence of dry substances bards of carbon sources other than glucose, which is assimilated by the yeast. The inventors showed that the dry matter bards contain about 10% formatiruem Sugars, mainly in the form of glucose or residual edge is small. Initial concentration of yeast corresponds approximately to 0.62 g of yeast per liter of medium, and the final concentration corresponds to approximately 13.8 g of yeast per liter of medium. Thus, in an environment formed around 13,2 g yeast per liter of medium, which corresponds to a consumption of approximately of 26.4 g of glucose. However, the initial glucose concentration in the flask No. 4 is about 11.2 g per liter of the medium. Thus, the amount of yeast is associated with a carbon source other than glucose, for example, with the presence of glycerol in the bard.

Of course, the present invention is not limited to the described and represented by way of implementation, which are given as illustrative and non-limiting examples.

1. The use of nutritional supplements derived from wheat bran fermented mold fungi, active beginning of which is at least one enzyme and a mixture of nutritious ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids, during simultaneous saccharification-fermentation to reduce the time this stage, growth and efficiency of yeast in the production of ethanol from starchy raw materials.

2. The use according to claim 1, where the nutritional Supplement is administered at this stage of the calculation 4-60 kg of dry matter per 1 ton of starch.

3. When is the change according to claim 1, where the nutritional Supplement is administered in solid or liquid form.

4. The use according to claim 1, where the nutritional Supplement has the following enzymatic activities:
- at least 500 units glucoamylase activity per gram of dry matter,
- at least 100 units of proteolytic activity per gram of dry matter,
- at least 100 units of xylanase activity per gram of dry matter,
and obtained by the fermentation of wheat bran mold fungi selected from strains of Aspergillus niger ATCC 201202, ATSS 76060, ATSS 76061, MUCL 28815, MUCL 28816. NRRL 3112 or strains of Aspergillus oryzae ATCC 22788 and ATSS 42149.

5. The use according to any one of claims 1 to 4, where a nutritional Supplement obtained through a process comprising the following stages:
i) providing wheat bran,
ii) hydrating, acidification to pH 4 to 5 and a temperature treatment of the bran so that pasteurized or sterilized them,
iii) sowing received wheat bran strain of Aspergillus niger, selected from among strains of ATSS 201202, ATSS 76060, ATSS 76061, MUCL 28815, MUCL 28816, NRRL 3112 or strain of Aspergillus oryzae, selected from among strains of ATSS 22788 and ATSS 42149,
iv) fermentation of wheat bran in solid state fermenter with periodic stirring at a temperature of from 28° to 38° C with a humidity of 45% to 65% by weight in terms of aeration sufficient to prevent constant is wow accumulation of carbon dioxide in the thickness of the wheat bran until when the fermentation will have the following minimum values of enzyme activity:
- glucoamylase: at least 500 units glucoamylase activity per gram of dry matter and, if
- proteolytic: at least 100 units of proteolytic activity per gram of dry matter,
- silananda: at least 100 units of xylanase activity per gram of dry substance.

6. The use according to claim 4, where the heat treatment stage (ii) is preferably carried out after wetting.

7. The use according to claim 1 or 4, where glucoamylase activity nutritional supplements is at least 750 units glucoamylase activity per gram of dry substance.

8. The use according to claim 1 or 4, where glucoamylase activity nutritional supplements is at least 1500 units of glucoamylase activity per gram of dry substance.

9. The use according to claim 1, where the vitamins in the nutritional Supplement are for vitamin C.

10. The use according to claim 1, where in the nutritional Supplement one or several amino acids are: alanine, arginine, asparagine, aspartic acid, valine, leucine, glutamic acid.

11. Method for the production of ethanol from a carbohydrate material which is a starchy raw material, including:
a) after the transition of the starch in the liquid phase and optional preliminary stage is shareware stage simultaneous saccharification and fermentation environment saccharification-fermentation glucose which in the early stages of simultaneous saccharification and fermentation is not more than 95% of the glucose equivalent, corresponding to the amount of starch in the above-mentioned starchy raw materials, and environment saccharification-fermentation are present in yeast as agent for alcoholic fermentation;
at this stage Wednesday saccharification-fermentation impose nutritional Supplement derived from wheat bran fermented mold fungi, active beginning of which is at least one enzyme and a mixture of nutritious ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids,
b) stage collecting ethanol, obtained in stage a).

12. The method according to claim 11, in which the glucose content in the medium in the early stages of simultaneous saccharification-fermentation is not more than 3% of glucose equivalent, corresponding to the amount of starch in the above starchy raw materials.

13. The method according to claim 11, where the nutritional Supplement is administered at this stage of the calculation 4-60 kg of dry matter per 1 ton of starch.

14. The method according to claim 11, in which stage simultaneous saccharification-fermentation is completely carried out without air or oxygen.

15. The method according to claim 11, in which stage simultaneous saccharification-fermentation includes an initial aerobic phase.

16. Pic is b according to item 11, in which stage of saccharification-fermentation is carried out in the following conditions:
- temperature: 30°C to 35°C,
- pH: lead in the early stages of simultaneous saccharification-fermentation up to a level of from about 3.5 to 5,
- inoculate yeast: approximately 106up to 5·108CFU of yeast per ml medium saccharification-fermentation,
the solids content from 20% to 35%,
the time of interaction: from 20 to 72 hours

17. The method according to claim 11, further comprising the final stage of the separation and release of dry bards/grains and liquid bards.

18. The method according to 17, in which alcoholic fermentation using yeast, which is obtained through the production of yeast under aerobic conditions in the fermenter prior to fermentation, and add them to the specified fermenter preliminary fermentation of clarified liquid Barda and/or concentrated Barda obtained by carrying out the method of production of ethanol through fermentation of carbohydrate raw materials, which was introduced a nutritional Supplement referred to in any of claims 1 to 10, and in which the used liquid bard, obtained in the process of ethanol production.

19. The use of nutritional supplements derived from wheat bran fermented mold fungi, active beginning of which is at least one enzyme and a mixture of nutritious ingredients for yeast: ergo terol, N-acetylglucosamine, vitamins, nucleic acids and amino acids, activation of alcoholic fermentation or pre-fermentation intended for preparation of yeast.

20. Method for the production of yeast under aerobic conditions in the fermenter prior to fermentation, characterized in that the said fermenter prior to fermentation add the clarified vinasse and/or concentrated vinasse obtained in the process of ethanol production through fermentation of starchy raw materials, which introduce a nutritional Supplement derived from wheat bran fermented mold fungi, active beginning of which is at least one enzyme and a mixture of nutritious ingredients for yeast: ergosterol, N-acetylglucosamine, vitamins, nucleic acids and amino acids.

21. The method according to claim 20, characterized in that the liquid vinasse is produced in the process in the implementation of the method according to 17, or 19.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: host cell Trichoderma reesei contains polynucleotides that code a heterologous polypeptide GH61, fusion protein of beta-glucosidase, cellulolytic ferments. The method to produce the composition includes cultivation of the host cell Trichoderma reesei and extraction of the composition. The composition conatins the polypeptide GH61, fusion protein of beta-glucosidase and one or more cellulolytic ferments. The method to decompose a material that contains cellulose is characterised by treatment of the material with a cellulolytic protein composition. The method to produce a fermentation product includes saccharification of the material, fermentation of the saccharified material with fermenting microorganisms and extraction of fermentation products.

EFFECT: presented inventions may be used for decomposition or transformation of a cellulose-containing material.

12 cl, 14 dwg, 23 ex

FIELD: biotechnology.

SUBSTANCE: alcohol (8-C) strain Saccharomyces cerevisiae No 8 having a high generative activity was deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM B-3855 and can be used in production of alcohol.

EFFECT: invention enables to increase the alcohol yield and to reduce the formation of byproducts.

3 tbl

FIELD: chemistry.

SUBSTANCE: method of fermenting low-molecular weight sugar to ethanol involves mixing low-molecular weight sugar, one or more fermenting microorganisms and modified biomass, fermenting the low-molecular weight sugar in conditions that are suitable for converting sugar to ethanol. The modified biomass has bulk density of less than about 0.5 g/cm3 and contains cellulose fibres that have been substantially irradiated and contain carboxylic acid groups. The fermenting microorganism includes yeast selected from a group consisting of S. cerevisiae and P. Stipitis or Zymomonas mobilis bacteria.

EFFECT: invention enables to obtain ethanol with output of at least 140%.

17 cl, 40 dwg, 78 tbl, 32 ex

FIELD: biotechnologies.

SUBSTANCE: method for simultaneous production of sugar and ethanol includes extraction of a sugar-containing juice from a suitable plant, simultaneous fermentation and treatment of the sugar-containing juice, simultaneous distillation of ethanol and concentration of the sugar-containing juice, crystallisation of the produced concentrate of the sugar-containing juice with production of sugar. Fermentation of the sugar-containing juice is carried out using strains of yeast Saccharomyces or Zygosaccharomyces, not producing a saccharose ferment, or a strain Saccharomyces cerevisiae BY4742 with a damaged gene of saccharase SUC2c, or a strain Saccharomyces cerevisiae Taiken 396 in presence of methyl-α-D-glucopyranoside.

EFFECT: conditions of fermentation make it possible to increase share of saccharose in a sugar syrup as a result of consumption in process of fermentation of sugars, which are not saccharose.

6 cl, 6 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing rectified ethyl alcohol and apparatus for realising said method. The method involves distillation of alcohol and attendant impurities in a mash column, purification of alcohol from head impurities in an epuration column, concentration of alcohol and purification thereof from intermediate impurities and residues of heat impurities in an alcohol column, and separation of impurities in a column for concentration of head and intermediate impurities. The wash distillate is fortified in the concentration part of the mash column; extraction of impurities in the epuration column is carried out with intense feeding of hot water for hydro-selection into the top tray and the middle part of the epuration column; purification of alcohol from methanol is carried out in a methanol column; lutter water from the alcohol column is fed into the top tray and the middle part of the column for concentration of head and intermediate impurities; lutter from the column for concentration of head and intermediate impurities is fed into the top tray and the middle part of the epuration column.

EFFECT: invention enables to obtain ethyl alcohol of high quality with minimum loss thereof with by-products.

2 cl, 1 tbl, 1 dwg

FIELD: food industry.

SUBSTANCE: method envisages raw material mixing with water at water duty equal to 1:(2.0-2.5), introduction of amylolytic enzyme preparations of diluent action in an amount of 0.5-1.0 units AS/g of reference starch, maintenance to produce a batch at a temperature of 65-70°C during 60 minutes with further maintenance at a temperature of 95-98°C during 60 minutes. Then the batch is cooled to 56-58°C, the amylolytic enzyme preparations of saccharifying action are introduced into the batch; final fermentative hydrolysis is performed during 30 minutes. The saccharified wort is cooled; a preparation of proteolytic action is introduced into the wort in an amount of 0.2-0.3 units PS/g of reference starch as well as seed yeast; the mixture is fermented during 48-54 hours. The raw material is represented by recyclable wastes of bakery production.

EFFECT: invention allows to simplify the ethyl alcohol production method, intensify the fermentation process combined with preservation of high quality characteristics of the final product and its yield increase.

2 tbl, 3 ex

FIELD: food industry.

SUBSTANCE: method envisages implementation of contact of a suspension containing granulated starch produced of vegetable raw material with α-amylase at a temperature below granulated starch gelation temperature. Oligosaccharides produced during this process are hydrolysed with the help of endogenic vegetable hydrolysis enzymes for production of wort containing more than 20% of glucose. Then the wort is fermented for subsequent production of ethanol from it.

EFFECT: invention allows to reduce energy intensity during ethanol production and increase ethanol yield.

20 cl, 1 dwg, 8 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: cellobiohydrolase polypeptide is selected from a group consisting of: a polypeptide containing an amino acid sequence having at least 95% identity to SEQ ID NO:6 and having cellobiohydrolase activity and an fragment a), having cellobiohydrolase activity. Such polypeptides can be obtained using a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention can be used in production of fermentable sugars and bioethanol from lignocellulose material via enzymatic conversion.

32 cl,15 dwg, 31 tbl, 32 ex

FIELD: food industry.

SUBSTANCE: vapour from the boiler is supplied into the bottom part of distillation column through the bubble flask. Wine material from the gravity vessel is supplied into the dephlegmator through a rotameter; the dephlegmator is represented by a heat exchanger with horizontal tubes wherethrough the wine material passes; alcohol-water vapours rising up through the distillation column are delivered into the intertube space; due to the vapours condensation the wine material heating is performed. The wine material heated up to 75-80°C is supplied, through the distribution device, into the distillation column and onto the nozzle ring with porcelain contact devices; during flowing down onto the nozzle ring with single-cap double boiling plates alcohol is completely removed from the material. Generated alcohol-water vapours rise up through the distillation column to the ring with multi-cap plates and consolidate due to interaction with phlegma flowing down through the distillation column. The alcohol-water vapours are dephlegmated and condensed in the dephlegmator and condenser. A part of condensate generated in the condenser in the form of heads draw is cooled in the refrigerator and supplied into the drain section. The other part of condensate is returned in the form of phlegma into the distillation column and onto the upper plate of the ring with multi-cap plates, alcohol-water vapours are sprayed and consolidated with this condensate. Condensate generated in the dephlegmator is cooled in the refrigerator to produce cognac alcohol with an alcoholic content of 62-70 vol. %. Slop generated in the distillation process is discharged from the distillation column blocking vapour outlet from it. The installation performance is 900-1000 dhal of absolute alcohol per day, vapour rate is 12-15 kg/dhal, pressure in the column bottom is 70-80 kPa. The produced cognac alcohol indices are the follows: colour - colourless; aroma and taste - without foreign smells and after-tastes; weight concentration of higher alcohols - 420 mg/100 cm3, medium esters - 90 mg/100 cm3, volatile acids - 40 mg/100 cm3.

EFFECT: invention ensures the process continuality, accelerates and simplifies the process due to the installation performance enhancement.

2 cl, 2 ex

FIELD: food industry.

SUBSTANCE: potatoes are thermally treated at a temperature of 0°C and below during from 20 days and longer. The potatoes are milled into mush and undergo fermentative saccharification. Optionally, potatoes thermal treatment at a temperature of 0°C and below may be combined with potatoes storage at a raw material storage facility.

EFFECT: invention ensures minimal energy expenditure, exclusion of sugars thermal destruction and melanoidin -generating reaction.

2 cl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining proteinase-activator of protein C in blood plasma includes cultivation of strains of Aspergillus ochraceus VKM F-4104D or Aspergillus ochraceus VKM F-4105D or Aspergillus ochraceus VKM F-4106D or Aspergillus ochraceus VKM F-4107D on nutritional medium at initial value pH 6.5. Nutritional medium contains (%): glucose 3.2-3.8, starch 0.1-1.0, fish flour hydrolysate 0.5-1.0, peptone 0.1-0.5, NaCl 0.1-0.2, KH2PO4 0.04-0.06, MgSO4-7H2O 0.04-0.06, water to 100%. Anticoagulant activity of solution of proteins of culture liquid of strains on elongation of activated partial thromboplastin time (CPTT) constitutes 1020-1032%. Activity of protein C activator in culture liquid of strains constitutes 79.8-89.9 units/ml.

EFFECT: increased activator activity.

2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from carbonate chernozem samples and deposited in Russian National Collection of Microorganisms, No. F-4106D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 960%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 77.9 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from plant residue samples on wort agar and deposited in Russian National Collection of Microorganisms, No. F-4105D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 920%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 72.5 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from soil sampled in Krasnodar Territory and deposited in Russian National Collection of Microorganisms, No. F-4104D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 960%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 77.5 units/ml.

EFFECT: higher proteinase activity of said strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: strain Aspergillus ochraceus is recovered from grey soil samples and deposited in Russian National Collection of Microorganisms, No. F-4107D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 955%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 76.1 units/ml.

EFFECT: higher activity.

1 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: pasteurised food product containing a proline-specific protease has water activity equal to at least 0.85. Used as the enzyme is protease extracted from Aspergillus or belonging to the S28 serine proteases family. The optimal activity of the said protease is at a pH value from 1 to 7, preferably - at a pH value from 2 to 6. Additionally proposed is a food product containing less than 1 wt % of protein or peptides. The said food products are produced by way of addition of a proline-specific protease to them.

EFFECT: such products consumption ensures gluten peptides splitting and is recommended to patients suffering from gluten intolerance.

17 cl, 5 dwg, 2 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to development of a novel strain used for preparing enzyme representing a complex of acid subacid proteases. Strain is prepared by selection from the known strain Aspergillus oryzae (VKPM F-683) by multistep selection using effective methods of mutagenesis. Strain is stored as lyophilic dried culture and on slants with wort-agar in the biotechnology section of enzyme preparations in the food processing department of the State Scientific Institute VNII of food processing technology in Moscow. Invention provides preparing the strain possessing the high level of synthesis of acid and subacid proteases and high total activity of enzyme in cultural fluid exceeding activity of analogue by 2.0-2.6-fold and in reducing culturing process time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-12 providing the high level of synthesis of acid proteases and xylanase. The strain is obtained by multistep selection of the strain Aspergillus oryzae-387 (VKPM F-683) using effective methods of mutagenesis. The strain is deposited in collection VKPM at № F-932. Invention provides preparing the enzyme complex showing the high proteolytic and xylanolytic activity in cultural fluid wherein their level exceeds activity in analogue by 2.5-2.8-fold.

EFFECT: valuable properties of strain.

2 tbl, 3 ex

FIELD: biotechnological methods.

SUBSTANCE: invention concerns new polynucleotides, which encode polypeptide having tripeptidylpentidase activity. To produce this polypeptide, host cell is transformed with polynucleotide or polynucleotide-containing vector and then cultured under suitable polynucleotide expression conditions.

EFFECT: enabled production of new protease from thread fungi.

14 cl, 3 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of biotechnology. Claimed is protein, including catalytic fragment of sialidase and possessing sialidase activity, which is selected from protein, whose sequence includes amino acids 274-666, or 274-681, or 290-666, or 290-681 SEQ ID NO:12, or includes amino acid sequence SEQ ID NO:14, described herein. Described is fused protein, which includes described above protein and anchor domain. Also given is information about molecules of nucleic acids, which code said proteins, expression vectors, containing said nucleic acids, and pharmaceutical compositions, containing said protein or fusion protein. Claimed is method of treatment or prevention of viral infection, caused by virus of influenza or parainfluenza, including application of therapeutically efficient amount of said composition to epithelial cells of subject.

EFFECT: invention makes it possible to extend arsenal of means against infection, caused by influenza or parainfluenza vitus.

25 cl, 2 tbl, 13 dwg, 17 ex

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