Enzymatically hydrolyzed wheat bran with glucoamylase, proteolytic and xylanase activity and method for their preparing by solid-phase fermentation of wheat bran using aspergillus niger

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.

EFFECT: improved preparing method, valuable properties of hydrolyzed bran.

14 cl, 10 tbl, 7 ex

 

The invention relates to enzymatic hydrolyzed wheat bran with glucoamylase, proteolytic and xylanase activities, as well as the way they are received by solid state fermentation of wheat bran by using Aspergillus niger.

Known to produce ethanol from corn starch by enzymatic cleavage, including stage liquefaction of starch with alpha-amylase to hydrolyze the starch with the formation of dextrins, next - stage saccharification using glucoamylase (otherwise called amyloglucosidase) for hydrolysis of dextrins with the formation of glucose and, finally, the stage of digestion of the latter to obtain ethyl alcohol.

The use of enzymes alpha-amylase and glucoamylase gives generally satisfactory results in cases when the source material take relatively pure starch milk, get wet milling of corn, but if you want to replace corn starch wheat starch or wheat flour, use only these two enzymes does not give acceptable results due to the presence of hemicellulose, which increase the viscosity ochrannych flour must so that the process becomes difficult. To overcome these difficulties it is necessary to use phase is sahariana special auxiliary enzymes. In addition, in the process of saccharification it is also desirable to use proteases to hydrolysis contained in the flour proteins, which will enrich the wort nitrogen before the next stage of alcoholic fermentation. Due to this it is possible to reduce the traditional provision of nitrogenous nutrition that is necessary for growth of yeast.

Although all these enzymes are available separately in the market in purified form, their disadvantage is the relatively high cost, leading to increased costs for obtaining ethanol from wheat. In addition, it is necessary to make the formulation of compositions on the basis of individual enzymes, which complicates the process.

Thus, there is a need for an inexpensive multienzyme preparation, which would combine glucoamylase, proteolytic and hemicellulase activity, in order to obtain ethanol from wheat flour with low cost.

The aim of the invention is to address this need.

The present invention relates to enzymatic hydrolyzed wheat bran with glucoamylase, proteolytic and xylanase activity, characterized by the fact that they spojeni using a strain of Aspergillus niger, and these glucoamylase, proteolytic and silananda activity have the following minimum values:

- glucoamylase at least 100 G per gram of dry matter,

- proteolytic - at least 100 P. units per gram of dry matter,

- silananda at least 100 units per gram of dry matter,

provided that glucoamylase activity should be at least 750 G per gram of dry matter and/or silananda activity should be at least 300 To units per gram of dry matter.

Preferably, glucoamylase activity was at least 1500 G per gram of dry matter and/or silananda activity was at least 400 units per gram of dry matter.

It is also advisable to proteolytic activity was at least 400 P. units per gram of dry matter.

The subject of the invention is also a method of obtaining an enzymatic hydrolyzed wheat bran, characterized in that it comprises the stages consisting in the fact that (a) take wheat bran; (b) moisturize specified bran and then subjecting them to heat treatment for the purpose of pasteurization or sterilization; (C) inoculant received wheat bran strain of Aspergillus niger; (g) when bran have the form of a layer thickness of at least 10 cm, produce their solid state fermentation reactor with aeration and periodic mixing for up to 3 days at a temperature of 28-38° With, preferably 32-36° s, and the initial moisture content in the specified bran set at 50-60 wt.%, supporting virtually unchanged during the time of fermentation, aeration conditions, preventing the accumulation of carbon dioxide, harmful effects on the fermentation reactor and due to the fermentation temperature increase above the recommended limits, until, until you have achieved the following minimum values of the enzymatic activity of the product of fermentation:

- glucoamylase at least 100 G per gram of dry matter,

- proteolytic - at least 100 P. units per gram of dry matter,

- silananda at least 100 units per gram of dry matter,

provided that glucoamylase activity should be at least 750 G per gram of dry matter and/or silananda activity should be at least 300 To units per gram of dry matter.

Preferably, glucoamylase activity was at least 1500 G per gram of dry matter and/or silananda activity was at least 400 units per gram of dry matter.

It is also advisable to proteolytic activity was at least 400 P. units per gram of dry matter.

It is advisable to choose the strain Asperillus niger from the group including strain NRLL 3112, strain ADS 76061 and strains derived from these strains through breeding or mutation in the need to achieve high glucoamylase activity. Particularly preferred strain of ATSS 76061.

If necessary, achieve high glucoamylase activity used as source material wheat bran should not be obankrochennye. Except for this limitation, you can use any type of bran, however, it is preferable that they contain a significant proportion (at least 40 wt.%) particle size of less than 1 mm.

Below as examples, do not have a restrictive nature, the characteristics of the two are suitable for these purposes bran.

FeaturesBran AndBran
Moisture (%)12,319,5
The protein content (% BB* )13,814,8
The starchy (% BB* )24,621,3
Particle size
>1.25 mm53,90,7
From 1.0 to 1.25 mm8,11,3
the t of 0.5 to 1.20 mm 33,368,2
From 0,25 to 0,5 mm3,724,6
From 0.16 to 0.25 mm0,32,6
<0.16 mm0,72,6
% CC - percentage in relation to the wet substance.

Wheat bran subject to moisture and heat treatment for the purpose of pasteurization or sterilization. It is preferable not to carry out a heat treatment to moisture, since in this order of operations were reported unsatisfactory results of digestion. Thermal processing may include, for example, heating in an autoclave. Very good results gave autoclave treatment for 20 minutes at a temperature 120-121° With, however, is quite acceptable and less hard modes (pasteurization in the drying chamber at a temperature of 105° C for 15 min). You can also perform heat treatment of the bran by the injection of water vapor in them that will allow simultaneously moisturizing bran.

It is advisable to maintain the pH value in the process of hydration in the range from 4 to 5.5, in order to enhance pasteurizing heat treatment and to facilitate the initiation of the fermentation process is necessary.

In addition to the sterilizing action of heat Oberburgermeister gelatinization contained in wheat bran starch, consequently, the availability of this substrate for the fungus Aspergillus niger, which allows a more efficient fermentation.

Essential hydration bran, as the moisture content influences the characteristics of the fermentation. First, the initial moisture content of the bran regulate the value of 50-60%, preferably 50-55%, of the total weight of bran and water, and support him in the fermentation process virtually unchanged in this range, for example, by periodically supplying water to compensate the loss of water from the environment. Saying "support virtually unchanged", we mean that it is acceptable to humidity took the values slightly deviate (±5% units) from the range of 50-60% over a relatively short period between two consecutive adjustments of humidity or at the end of fermentation. In any case, it is advisable that the humidity does not fall below 45%. Humidity culture nutrient medium demonstrates a decreasing trend in the growth process due to evaporation due to temperature increase caused by the growth of fungi, given that this environment is a poor conductor of heat. A significant role is played by the quality of the water used. You can use high-quality tap water or distilled water./p>

Inoculation of wheat bran can be made of any suitable inoculum. Specialists in this field there are many ways of making the necessary inoculum from a pre-selected strain. It is advisable that the amount of inoculum was, at least, 1× 107spores/gram of initial dry matter.

The fermentation may be conducted in any suitable reactor. The examples used for this purpose reactors are given in the article by A. Duran with co-authors, published in Agro-Food-Industry Hi-Tech (may-June 1997, p.39-42).

The fermentation can be carried out during the time from 1 to 3 days, preferably from 30 to 60 hours. When processing time less than 1 day, the fermentation is too incomplete. After 3 days fermentation is completed, or almost completed, so that its further uneconomical. The temperature of the medium support, usually in the range from 28 to 38° C, preferably from 32 to 36° S, which corresponds to the optimal range of activity observed in strains of Aspergillus niger, selected for the purposes of the invention. It is advisable to maintain the temperature of the air in the first hours of fermentation in the range of 34-38° With the purpose of stimulating germination of spores, and then reduce it to 28-32° in the remaining time of fermentation with the aim of Regulus is the formation temperature of the environment.

The pH value of the fermentation medium is usually not regulate. If the source is close to 6,0-6,4, in the process of growing it decreases to 3.8 to 4.2, and on its completion increases. These changes correlate generally with the phase of sporulation of the fungus. The change in pH gives a visual representation of the state of culture.

It is necessary to provide in the fermenter aeration, preferably continuous, in order to ensure a supply necessary for fermentation of oxygen and to prevent excessive accumulation of carbon dioxide produced during fermentation. In addition, aeration helps to regulate temperature and humidity culture medium. It is expedient to provide sufficient saturation of air with water in order to limit the tendency to dehydration environment. To give any quantitative information on the intensity of aeration quite difficult, as there are many variables play a role, in particular, as the size and configuration of the reactor, the number of downloaded bran, etc. However, by simple model experiments, the expert can easily determine the necessary in each case the flow.

Download bran in the reactor must be periodically replenished in the fermentation process using a special mixing machines such as levers, blade or spatula, or is of neki prevent the formation of an impenetrable mass and the to aeration affected the entire mass of bran evenly. It should, however, avoid too intensive mixing, as this may cause damage to the fungus.

Claimed according to the invention the product is a solid product that is suitable, in particular, to obtain ethanol from wheat. It can be added directly to the liquefied starch (dextrins), obtained at the stage of dissolution, in order to perform saccharification. Used as such, the most significant factor is glucoamylase activity. It is therefore advisable to use the product according to the invention, which would have glucoamylase activity, for example, not less than 750 G%, preferably not less than 1500 G per gram of dry matter.

Another possible use of the claimed product relates to the production of feed wheat based for monogastric animals (mono-gastric), such as poultry and pigs. In this case, the most important factor is silananda activity. So here it is advisable to use a product that would have xylanase activity, for example, not less than 400 units per gram of dry matter.

If necessary, the offered product can be subjected to drying or freezing for storage purposes.

Drying should be done p and moderate temperatures, so as not to cause adverse effects on enzymatic activity. For example, good results were drying in a drying chamber at a temperature of 40° C. Freezing can be applied to wet foods at a low temperature, for example, -20° C.

During the processes illustrated in the following examples, various enzymatic activity was measured using the following methods.

a) Glucoamylase activity

When exposed to drug glucoamylase (HA) starch solution is released reducing sugars. When heated to 100° in the presence of 3,5-dinitrosalicylic acid (BPS), these songs take brown determined by spectrophotometer (Kontron Instruments, Milan, Italy) at 540 nm.

The reaction medium contains:

- 1%starch solution 500 μl;

nitrate buffer 450 ál;

solution of enzyme 50 ál.

The reaction proceeds for 30 min at a temperature of 60° With (for drugs glucoamylase A. orizae - 55°). Every 5 min make sampling, mix them with the BPS and placed in an ice bath. After their heated for 5 min at a temperature of 100° C, rapidly cooled, and finally produce their quantitative analysis at 540 nm.

These terms of quantitative analysis were determined after studying the influence of those is temperature and pH on the activity of drugs HA. As a substrate for this enzyme hydrolysis used soluble starch Merck (Darmstadt, Germany). BPS prepared in accordance with the procedure proposed .Bernfeld, Methods in enzymology, 1, 149-159 (1955):

- pre-dissolve:

to 10 g of 3,5-dinitrosalicylic acid

- 200 ml of 2-molar sodium hydroxide

- 200 ml of distilled water

next to add:

- 300 g of the tartrate of sodium and potassium

- to bring the volume to 1 l with distilled water after complete dissolution.

After preparation of this reagent should be stored with protection from the action of light. Calibration curves constructed using glucose as the reference product for the quantitative analysis glucoamylase activity and control reactions liquefaction-saccharification and using xylose for measurement of xylanase activity.

One glucoamylase activity (G unit) corresponds to the amount of enzyme necessary to break down one micromole of substrate per minute, where as the standard uses glucose. Glucoamylase activity, calculated by the formula below, are correlated with the number of initial dry matter (ICB):

A=(R/V)*(V/Mperm)

- A - HA activity, expressed in G units· g ICB-1(µmol· min-1·g ICB-1),

- R the rate of excretion of glucose equivalents in µmol· min-1,

- V - volume of enzyme solution, defined in ml,

- V - total volume of distilled water used for the extraction of enzyme solution, in ml,

- Mperm, expressed in g ICB, is the initial weight of the dry product from which the extracted enzyme solution.

b) Proteasa activity

This quantitative analysis was developed for azocasein by the method of Banana described in "protein purification Methods - a Practical approach" edited by ALL of Harris and C. Engel, IRL-Press, Oxford University Press, 1-66 (1989). Cleavage of this substrate by the protease leads to the selection of isogroup that absorb in the UV-range of 340 nm. Change of absorption in the process of the kinetics of hydrolysis of the protein is possible to judge the intensity of the reaction.

The reaction medium contains:

- 1%-aqueous solution of azocasein with pH 5.0 1000 ál

solution 200 ál of enzyme

Azocasein (Sigma, St. Louis, USA) dissolved in acetate buffer with 0.1 M pH 5.0. Quantitative analysis protease activities conducted at the same pH, because at lower values azocasein insoluble in the specified acetate buffer. The enzymatic reaction is carried out at a temperature of 60° C. for 20 min every 5 min make sampling and mixing them with 5%trichloroacetic acid (THU) to terminate the reaction.

On the on the unit by activity (P units) corresponds to the number of enzymes, necessary to increase the unit AndNm0.01 per minute, is caused by the release of isogroup in the above conditions. This activity calculated by the formula below, are correlated with the original dry matter (P%· g-1ISV) or glucoamylase activity (P%· g unit-1):

A=(R/V)· (V/Mperm)

And proteasa activity, expressed in N units· g ICB-1,

- R - the rate of emission of isogroup expressed in the form of increased unit AndNmmin-10.01,

- V - volume of enzyme solution, defined in ml,

- V - total volume of distilled water used for the extraction of enzyme solution, in ml,

- Mperm, expressed in g ICB, is the initial weight of the dry product from which the extracted enzyme solution.

in) Silananda activity

To detect this enzyme activity was carried out by reaction of drugs HA with a solution of soluble xylan and measured the amount of the reducing sugars by the method of BPS.

The reaction medium includes:

- 1%-aqueous solution of xylan with pH 4.5 900 ál

- enzyme solution 100 ál

The solution larch xylan (gum) (Sigma, 1%) prepared in citrate buffer pH 4.5 and conduct the reaction at a temperature of 60° C. for 20 min every 5 min take samples, mix the x with BPS and placed in an ice bath. Then produce their quantitative analysis in accordance with the procedure similar to that which was described with reference to the measurement of HA activity using xylose as standard.

One unit of xylanase activity (units) corresponds to the number of enzymes required to select one of micromole reducing sugars per minute. This activity correlated with the original dry matter (%· g-1ISV) or glucoamylase activity (units· G ed-1). To calculate this activity, use the same formula for the calculation of HA-activities, where:

And silananda activity, expressed in units· g ICB-1(µmol· min-1·g ICB-1),

- R - the rate of excretion of xylose equivalents in µmol· min-1,

- other members of the formulas remain unchanged.

EXAMPLE 1 - the Selection of strains Asperyllus

Conducted a comparative study of the ability of seven different commercially available strains of Aspergillus to produce glucoamylase during solid state fermentation of wheat bran.

Experiments were performed on 50 g of fermentation medium in Erlenmeyer flask. The environment consisted of 21.5 g of wheat bran, 27.5 g of water and 1 g of wheat starch. The initial value of pH ranged from 6.0 to 6.5. The medium was sterilized in the autoclave for 20 minutes at a temperature of 120° is.

Every Wednesday sowed the test strain in the amount of 2× 107spores per gram of initial dry matter. Age dispute was equal to 3 days. Provided the ability to ferment for 40-50 hours, the Erlenmeyer flask was placed in a thermostat with a temperature of 35° C. Upon completion of the fermentation, the fermented medium was stirred with 150 ml of distilled water to translate the obtained enzyme solution, after which the mixture was subjected to filtration to extract the enzyme solution. The solution was centrifuged to remove residual spores and particles, then Packed up the solution in the 100 ml vials, which were stored at -20° to determine glucoamylase activity.

The tested strains and the results obtained are summarized in the following Table 1.

Table 1
Reference strains from collectionsThe duration of the solid-phase fermentation of an hour.HA activity (G unit· g-1ISV)
A.niger ATCC 7606050627
A.niger ATCC 7606150943
A.niger MUCL 2881540710
A.niger MUCL 2881640631
A.niger NRRL 311250 1056
A.oryzae ATCC 2278850903
A.oryzae ATCC 4214950861

You may notice that the A.niger strains NRLL 3112, A.niger ATCC 76061 and .oryzae ATCC 22788 exhibit the highest activity in terms of production glucoamylase.

However, you should take into account another important property is the stability of the resulting glucoamylase. Therefore, tests were carried out on thermal resistance by heat treatment of the enzyme solution at a temperature of 55 and 60° C for 30 min measurement after this time glucoamylase activity. These operations are similar to those operating modes used in the saccharification of starch. It was found that the most stable

glucoamylase received from A.niger strains ATCC 76061 and A.niger NRLL 3112 (100%residual activity after 30 min at a temperature of 55° and about 50%residual activity after 30 min at a temperature of 60° C), whereas strains of A.oryzae ATCC 22788 and ATCC 42149 give glucoamylase with zero residual activity after 30 min at a temperature of 60° and 46%residual activity after 30 min at a temperature of 55° C. Therefore were selected it strains Apeg ATCC 76061 and NRLL 3112. In addition, it was found that the strain Apeg NRLL 3112 genetically quite unstable (loss of activity after several what their reproductive cycles), so the most preferred should recognize all same strain Apeg ATCC 76061. For this reason he was chosen for further examples.

EXAMPLE 2 - Getting glucoamylase in non-sterile experimental tanks, 50 l, supplied by INRA: the importance of pre-treatment of wheat bran

Experiments were performed using non-sterile fermenter with a capacity of 50 l, similar to that described in the paper cited above article Adurna with co (figure 1), and wheat bran brand ALL supplied alcohol plant Brie Champagne Ethanol in Provins, France. To obtain 5 kg of culture medium with 55%moisture content used two methods of cooking bran:

dry bran: bran is sterilized in an autoclave for 1 h at a temperature of 105° C, then mixed with water (experience F4C3);

wet bran: bran moistened in a kneading machine to 45% and autoclave for 20 min at a temperature of 121° With (experience F4C4).

In both cases produce the inoculation amount equal to 2× 107dispute· g-1ST (dry matter), and regulate the moisture content of the media on the value of the order of 55%. They are then fermented to 10-cm layer in aerated tanks. During these operations, the growing medium is subjected to intermittent raseiniu spatula to reduce its temperature. During fermentation the atmosphere is the fer continuously update an air-conditioned temperature, humidity and flow rate which are given in tables.

The results are summarized in Table 2 (for experience F4C3) and 3 (for experience F4C4).

The obtained data allow to draw the following conclusions.

- To ensure efficient production of glucoamylase prior heat treatment of wheat bran to perform their hydration. In addition to disinfection, heat treatment of wheat bran contributes probably the gelatinization of starch.

The pH value seems to be a good quantitative indicator of the dynamics of the growth and production of mushroom HA, not allowing, however, to evaluate the obtained amounts of HA.

- Moderate stirring (rastekanie) environment does not exert any harmful effects on the production of enzymes.

- Change of moisture content in the process is described cycles of fermentation indicates significant drying of the culture medium, which can harm the growth of fungi.

EXAMPLE 3 - Getting glucoamylase in experimental fermenter supplied by the company FUJIWARA: the importance of maintaining the humidity of the environment in the process of fermentation

This experiment was carried out using a fermentor, manufactured by FUJIWARA, Okayama, Japan, and wheat bran brand EVERYTHING. It differs from the fermentor as described in Example 2, is, in particular, in diameter Chan, to whom that is equal to 0.66 m, unlike 0.35 m for vats INRA. This fermenter is necessary to use 20 kg environment with 55%water content, prepared by the "wet method of bran as described in Example 2, which gives the possibility of growing the layer thickness of 12 see Mixing is provided by three continuously rotating vertical screw conveyors that shake up the environment in a rotating Chan (5-10 min.). In the fermentation process, as in the case with vats INRA in Example 2, the gas atmosphere is continuously replaced by the conditioned air temperature, humidity and flow rate which are given in Table 4.

During this experience, which is assigned the index FII, explored the possibility of regulating the moisture content. Here resorted to a point measurements of humidity culture using an infrared device, as well as mass medium to determine the necessary amount of water added to maintain the moisture content of the environment at the level of more than 50%.

The results of this experience FII are summarized in Table 4. The obtained data call for the following comments.

From experience FII should be pretty obvious that by maintaining the moisture level of 50 to 55% stimulates the production of enzymes emitting 1600 units/g WW after 44 h of fermentation, more than double the activity achieved during the experience F4C2 in Example 2, where similar regulate the tion is not provided.

- Stabilization of enzyme production after 44 h of fermentation, coinciding with the emergence of fungal spores, indicates that there is no need to continue growing at the end of this phase.

Satisfactory temperature control medium at about 35° can be achieved by the proper combination of air conditioning with mixing environment.

Culture freely withstands intermittent stirring, by appropriate means, provided in the fermenter firm FUJIWARA.

EXAMPLE 4 - Receive glucoamylase in experimental fermenter INRA capacity of 50 l with agitator: a preliminary feasibility steam treatment of the bran and cultivation in the "sterile conditions"

This pilot fermenter is similar to that described in document WO-A-9418306 and shown in figure 4 in the paper cited above article A. Duran with co-workers. With this setting it becomes possible steam treatment bran directly in the fermenter, which is the method of preparation on an industrial scale is preferred. In addition, culture is prepared, inoculated and grown in sterile conditions, with the exception of sampling, which gives the experience polyarylenes character, unlike the previous two examples.

(A) conditions of experiment

<> In the fermenter load 9 kg bran ALL, previously moistened with water in the amount of 1.5 l, and then sterilized directly on the spot for 20 minutes at a temperature of 121° with periodic mixing for 5 seconds every 5 minutes. Due to such processing, it is possible to achieve moisture content of 46%, which subsequently, at the stage of inoculation, lead up to 55%.

Bran inoculant drug type koji:

180 g ALL bran (source humidity 55%), fermented for 4 days at a temperature of 35° C, mixed with 3 liters of sterilized water to obtain a suspension of spores, part of the inoculate.

The initial conditions of fermentation are:

18.3 kg of culture with 55%humidity and the initial pH of 5.7;

the layer height 40 cm;

the rate of aeration 314 l· min-1;

the temperature of the incoming air 35° C;

the relative humidity of the incoming air 95%.

B) Monitoring of fermentation

In addition to the determination of pH, temperature, percentage dry matter content and production of the HA, perform continuous registration of change of mass culture in a 50-liter fermenter with a stirrer, and under non-sterile reactor produce weighing culture after 21 h and 42 h of fermentation.

Such measurements of the masses have a twofold benefit.

Maintaining moisture in the growing process through which the your estimate of the percentage of dry matter (DM)

In the fermentation process to reduce mass culture contribute to two factors, namely:

- the drying medium, which is compensated through the water inlet;

- loss of dry matter, which is associated with the growth of the fungus.

This loss of dry matter quite substantial: for 40 h of cultivation lost 20% of ST, or 0.5% SV per hour, assuming in order approximation that the losses are linear.

On this basis, if known mass culture (M(t)in a certain point in time, it is possible to deduce from it theoretical percentage of ST at time t according to the following formula:

where ICB is the number of initial dry matter.

When calculated in this way % ST exceeds 50%, add sterilized water to reduce to 45%.

Results per gram of the original ST.

Changes in mass and measured the percentage of SV allow you to calculate the real loss of dry matter (LStexpressed in %) in the growth process. Thus, the number of HECTARES, which is still expressed in G units· g-1SV can be expressed in G units · g-1the original SV by using the following formula:

(G unit· g-1ICB)=(G unit· g-1ST.)· (100-LSt)/100

In the Results

In Tables 5 and 6 summarizes the operating conditions and results obtained in the eastore with stirrer subjected to steam treatment of the bran.

Despite the aeration of the culture water saturated air, it is dehydrated so that had, as can be seen from Table 5, twice to adjust its moisture content, when it fell to less than 50%.

Managed to maintain the temperature of the medium at the secondary level 35° by reducing the supplied amount of air, but mainly with intermittent mixing.

In these culture conditions, the growth of the fungus whose movement was controlled by measuring the pH is maintained for 60 hours and allows you to achieve production 1436 units· g-1ST. during 44 h and 1990 units· g-1ST. 63 hours If you give the amounts of HA to the original SV, it will constitute, respectively, and 1540 1160 G unit· g-1ICB. For comparison, in the framework of Example 3 for 44 h of cultivation was obtained 1605 units · g-1SV, which is equivalent 1067 G unit · g-1ICB. You can mark certain benefits, because the data show that although in both experiments, the performance is the same, but the conditions of the experiment of Example 4 to give the opportunity to extend the production of enzymes even at the height of the layer 40, see

Thus, the processing bran steam with subsequent digestion in 50-liter reactor INRA with agitator extends a growing mushroom enzyme production.

If Pref is STI the amounts of HA to the original ST., it will be 1540 G unit · g-1ICB.

For the same samples was carried out quantitative determination of xylanase and by activities. The results obtained were quite satisfactory, with maximum values after 50 h of fermentation averaged:

350 To unit· g-1ISV for xylanases;

400 P%· g-1ISV for proteases.

Due to the continuous registration of the masses was possible to calculate the loss of dry matter in the growing process. She was about 23% after 60 h of cultivation (up to 2% taking into account the error weighting).

EXAMPLE 5 - Using the fermented bran obtained in the experiment Example 4, for hydrolysis of wheat flour

Conducted a series of experiments on saccharification bran obtained in Example 4, wheat flour, previously subjected to traditional enzymatic liquefaction. As a control sample used glucoamylase drug AMG 300L®, manufactured by NOVO. The object of these experiments was the usual wheat flour type 45. Operating modes are summarized in Table 7 with regard to the number wort 750,

Table 7
ProductAMG 300L® (Novo)Fermented branDried fermented bran
AMG 300 LExample 4Example 4
LiquidWet branDry bran
The method of storageat +5°at -20°at room t-d
FlourCommercial type 45Commercial type 45Commercial type 45
The amount used (g)300300300
Dry chemical environment (%)353535
Conditions liquefaction1 h,88°, rn,11 h, 88° C, pH of 6.21 h, 88° C, pH of 6.2
Enzyme125 ál termopilas 120L®125 ál termopilas 120L®125 ál termopilas 120L®
Conditions of saccharification44 h, 58° C, pH 4.640 hours, 58° C, pH 4.5544 h, 60° C, pH 4.52
Qty, equiv. 3500 G unit205 vrk4.3 g2.1 g

During this hydrolysis of wheat flour each time produced a selection of three samples of the environment. Results the concentrations of reducing sugars (PC) at different points in saccharification, are shown in Table 8, represent the Wallpaper averages for these three selections. These quantitative determination, for which the technique was applied BPS, were performed on the supernatant liquid was centrifuged samples. We measured also the final viscosity ochrannych products.

Table 8
Measured valueAMG 300L® (Novo)Fermented branDried fermented bran
Concr (source (g/l))180,9±4,6to 185.0±3,1which is 171,5±4,5
Final conc. PC

(g/l)
327,5±18,5325,0±22,5348,3±19,1
Viscosity (MPa·)to 6.802,822,80

In addition, there has been an increase in the content of soluble nitrogen in wort after saccharification, which is explained by the proteolytic action of fermented bran.

These results show that obtained in the experiments in Example 4 fermented bran is able to hydrolyze wheat flour as effective as the standard drug HA, regardless of the method of storage.

Hydrolysis of the fermented flour bran also leads to a substantial decrease in viscosity compared to conventional enzyme preparation.

EXAMPLE 6

This example illustrates the possibility of the ability to get significant amounts of xylanase and a small number of glucoamylase using a strain of Aspergillus niger.

Consider the experiment was carried out on the experimental fermenter firm Fujiwara using ALL bran and strain A. niger ATCC 201202, known for its ability to produce xylanases. The experimental work of the fermenter as described in Example 3. In this case took 20 kg environment with 55%humidity, prepared as described in Example 2. As in Example 3, the ambient humidity was maintained in the fermentation process at the level of more than 50% and regulate the temperature of the medium within about 35° C.

After 37 hours of fermentation under these conditions the strain A. niger ATCC 201202 made of fermented bran containing 727 To unit· g ST and 162 G unit· g ST.

EXAMPLE 7 - the inclusion of fermented bran according to the invention in birdseed wheat based, designed for broilers

It is known that contained in wheat flour hemicellulase partially soluble in water and increase the viscosity of the contents of the intestine, resulting in attenuated the secretion and absorption of nutrients.

It was found that adding hemicellulase leads to the decomposition of hemicellulose, which makes it possible to reduce the viscosity of intestinal contents and improve the zootechnical performance of monogastric animals such as broilers fed forage only slaco is in the composition of which is wheat.

Experiment was carried out at 1200 broilers Ross to demonstrate the benefits of fermented bran with hemicellulase (xylanase) activity in comparison with betterment food and feed, containing a standard source of xylanase - product Avizyme®. Feed enzyme or without prepared thus to feed 4 groups of 300 chickens. The detailed composition of this feed are shown in Table 9. In the first 21 days of feeding the broilers were given food for growth (KR), which was then replaced by "finishing" food (DK) for the next 18 days.

Table 9
FoodCUDK
Humidity (%)10,611,4
Protein (%)21,319,1
Fatty substances (%)6,16,4

Feed 1 enzyme was added. In feed 2 and 3 were, respectively, 3 and 5 kg of fermented bran per ton of feed. To feed 4 consisted of 0.6 kg Avizyme® per ton of feed.

The results of this experience after 39 days of feeding are summarized in Table 10.

Table 10
Food1234
Fermented bran according to the invention (kg/tonne)and-3,05,0-
AVIZYME® Finfeed (kg/tonne)b---0,6
Silananda activity (units/kg feed)-170028401620
The ratio of feed through 39 daysin1,7751,7481,7381,745
Reduction in the rate of transformation. feed (% of feed 1)g-1,522,081,69
Mortality (%)2,32,03,02,3
A. these fermented bran showed glucoamylase activity equal to 1000 G unit/g dry matter, proteolytic activity 125 P units/g dry matter and xylanase activity 600 K units / g dry matter;

B. Avizyme® comes Finnish company Finfeed;

C. the ratio of the weight of consumed food for weight gain

year percentage decrease in the weight of the consumed feed relative to weight of feed consumed 1 (without enzyme).

Thanks to the inclusion of fermented bran in birdseed (3 or 5 kg/ton) was able to significantly reduce the coefficient of transformation the feed. In the experiment using a dose of fermented bran of more than 3 kg/tonne does not, apparently, any significant practical benefits. Marked improvement comparable to that obtained when using a commercially available product Avizyme® (0.6 kg/tonne). However, the use of fermented bran has the advantage that it is less expensive than working with an enzyme product available on the market.

Of course, the above-described embodiments of the invention are given only as examples, so that they may be various modifications, in particular by substitution of technical equivalents, if you do not take this beyond the scope of the invention.

1. Enzymatic-hydrolyzed wheat bran with glucoamylase, proteolytic and xylanase activity, characterized in that they are enzymatically hydrolysed by the microorganism Aspergillus niger, and these glucoamylase, proteolytic and silananda activity have the following minimum values: glucoamylase at least 100 G per gram of dry matter; proteolytic - at least 100 P. units per gram the dry matter; silananda at least 100 units per gram of dry matter, provided that glucoamylase activity is at least 750 G per gram of dry matter and/or silananda activity is at least 300 To units per gram of dry matter.

2. Enzymatic-hydrolyzed wheat bran according to claim 1, characterized in that glucoamylase activity is at least 750 G per gram of dry matter.

3. Enzymatic-hydrolyzed wheat bran according to claim 1, characterized in that glucoamylase activity is at least 1500 G per gram of dry matter.

4. Enzymatic-hydrolyzed wheat bran according to claim 1, characterized in that silananda activity is at least 300 To units per gram of dry matter.

5. Enzymatic-hydrolyzed wheat bran according to claim 1, characterized in that silananda activity is at least 400 units per gram of dry matter.

6. Enzymatic-hydrolyzed wheat bran according to claim 1, characterized in that the microorganism Aspergillus niger selected from the group comprising a strain of Aspergillus niger NRLL 3112 and a strain of Aspergillus niger was ATSS 76061.

7. Enzymatic-hydrolyzed wheat bran according to claim 6, characterized in that the microorganism Aspergillus niger is a strain of Aspergillus niger was ATSS 76061.

8. Enzyme-gidralizovanny the wheat bran according to any one of claims 1 to 7, characterized in that they are used for the production of ethanol from wheat.

9. Enzymatic-hydrolyzed wheat bran according to any one of claims 1 to 7, characterized in that they are used as an additive to feed monogastric animals.

10. The method of obtaining enzymatic-hydrolyzed wheat bran according to claim 1, characterized by the fact that wheat bran moistened, then these bran is subjected to heat treatment for the purpose of pasteurization or sterilization, inoculant wheat bran obtained by the microorganism Aspergillus niger, make solid-phase fermentation of bran in a layer thickness of at least 10 cm in the reactor with aeration and periodic stirring for 1-3 days at a temperature of 28-38°s, and the initial moisture content in the specified bran set at 50-60 wt.%, supporting virtually unchanged during the time of fermentation, aeration conditions, preventing the accumulation of carbon dioxide, to achieve the following minimum values of the enzymatic activity of the product of fermentation: glucoamylase at least 100 G per gram of dry matter; proteolytic - at least 100 P. units per gram of dry matter; silananda at least 100 units per gram of dry matter, provided that glucoamylase activity costs is t, at least 750 units of Grams per gram of dry matter and/or silananda activity - at least 300 units To per gram of dry matter.

11. The method according to claim 10, characterized in that the microorganism Aspergillus niger selected from the group comprising a strain of Aspergillus niger NRLL 3112 and a strain of Aspergillus niger was ATSS 76061.

12. The method according to claim 11, characterized in that the microorganism Aspergillus niger is a strain of Aspergillus niger was ATSS 76061.

13. The method according to claim 10, characterized in that the fermentation process does not allow for the drop in humidity bran less than 45%.

14. The method according to claim 10, characterized in that it comprises an additional step consisting in freezing or drying the obtained product.



 

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