Strain aspergillus niger-producer of citric acid

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The strain of fungus Aspergillus niger B-6 is the producer of citric acid. The strain Aspergillus niger is deposited in the joint Russian Collection of Agrocultural Microorganisms RAAS (RCAM) under registration number RCAM02149 and can be applied in the production of citric acid.

EFFECT: invention makes it possible to increase the output of citric acid.

2 tbl, 6 ex

 

The invention relates to the microbiological industry and relates to the breeding of a new strain of the fungus Aspergillus niger is the product of citric acid for deep fermentation method, Saharsa-mineral media and environments, hydrolysates prepared from starchy raw materials.

Known strain of micromycetes Aspergillus niger F-696 (Patent RF №2078810, 1997), which is aged in citric acid, Saharsa-mineral nutrient medium and medium prepared from hydrolyzed starch. However, the yield of citric acid from sugar when using this strain in deep conditions is not high enough and saharso-mineral environment 87,6%. Strain aged hydrolysates of starch technology with the introduction of additional sugars in the fermentation process, resulting in an increase of the amount of sugar per cycle that affects the yield of citric acid, 82.8 per cent.

As a prototype the selected known strain of micromycetes Aspergillus niger F-501. (Patent RF №1811697, 1991), designed for deep cultivation on Saharsa-mineral environments. The disadvantage of this strain is the low intensity of biosynthesis of citric acid per unit volume of medium per day is 18.2 g/(DM3·day) and not enough high yield of citric acid from spent sugar - 82,2%.

The invention is aimed at obtaining a new genetically mustache�stable strain, possessing a higher activity of the biosynthesis of citric acid by fermentation of Saharsa-mineral media and media made from hydrolysates of different types of starchy raw materials.

When cultured on Saharsa-mineral environment in deep conditions forms a new strain of citric acid with the release from spent sugar 90,5%, which is significantly superior to the prototype strain VKPM F-501. When fermentation on media made from hydrolysates of starchy raw materials, in-depth conditions for 4.5-5 days of fermentation of a new strain produces citric acid with a sufficiently high yield from spent sugar and 84.7-99.3% of (table.1).

A new strain of micromycetes Aspergillus niger obtained from an Aspergillus niger strain VKPM F-171 using chemical mutagens (diethylsulfate) and under the action of irradiation of UV rays at a dose of 2.8 thousand erg/mm2in combination with the selection of resistant spontaneous variants.

New selected strain is deposited in the Departmental collection of useful microorganisms for agricultural purposes RAAS under registration number Cam system for true 02149.

The strain was identified by cultural, morphological (size and color of the colonies, type of mycelium and conidial heads) and physiological and biochemical (intensity of biosynthesis of citric acid on starchy media) properties�. The properties are presented in table 1.

The strain Aspergillus niger Cam system for true 02149 is characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural morphological traits

Cultural morphological traits identified in pachystachya crops grown on wort-agar medium.

Colony diameter on average 81 mm light beige color. Aerial mycelium well developed. In the center of the colony conidial heads are tight. From the center to the edge of the colony conidial heads are smaller. On the edge of the colony within a radius of 8 mm light area with immature white conidial heads, tightly pressed to the substrate mycelium. Reverse side of colony smooth white.

Conidial heads round shape with a diameter from 40 to 125 μm. Bloating of canadianese (vial) spherical shape ranging in size from 33 to 65 μm. Sterigma bilayer: the length of sterigma the first layer is from 12 to 44 microns, the second layer is from 10 to 16 μm. Conidia are round with a thick cream shell subulate. The average diameter of 4.5 µm conidia. Conidiophores erect, hyaline, their length - from 510 to 1410 microns, width from 11 to 22 micrometers.

The strain Aspergillus niger Cam system for true 02149 differs from the known beige strain F-501 smaller colonies (81 mm in contrast to 94 mm) and has a large range of morphological structures (length of canadianese, sterigma second layer, to�idii).

Physiological and biochemical characteristics

A new strain of Aspergillus niger Cam system for true 02149 is an aerobe, prototroph, has an optimal growth temperature of 32°C. the Temperature range of growth 28-36°, the optimum pH 1,7-7,0.

The sources of nitrogen

Assimilates nitrogen organic compounds (peptone, protein, amino acids, yeast autolysis, mycelial mass, urea), assimilates the mineral nitrogen compounds, ammonium salts, nitrates.

The sources of carbon

Well assimilated carbohydrate-containig sources and grows on molasses, saccharose, glucose, starch hydrolysates, hydrolyzed flour from grain (rye, rice) and hydrolysates of cereal milling (rye, wheat, rice), containing glucose, maltose, dextrins.

The new strain is stored in the form of air-dry conidia with a residual humidity of no more than 10% at a constant room temperature and relative air humidity not exceeding 75%. Not be exposed to direct sunlight.

Compared to the known strain strain Cam system for true 02149 when the fermentation Saharsa-mineral and starchy raw materials synthesizes much more citric acid (see table 2).

Summary of the invention illustrate specific examples of the use of Cam system for true 02149.

EXAMPLE 1

For laboratory tests seeds are grown in test tubes on the sloped wort-agar. For p�suprainfection and production testing of seeds grown in the ditches on a dense nutrient medium, prepared on the basis of neohmelennoe beer wort containing 7% dry matter, and compacted agar-agar (20 g/DM3) pH of 5.8. The height of the layer of the medium 12-13 mm.

The sterile medium was inoculated with conidia of culture of the fungus and incubated in an incubator at 32°C for 7 days. The relative humidity of the air in the heat chamber is gradually reduced: the first four days - 80% -60%, the last three days - 60% -40%. After 7 days conidia removed from the surface of the mushroom tapes in cuvettes using a special vacuum device. For full ripening and drying in vitro culture of the fungus incubated at room temperature for 2 days. Wet conidia with a ditch dried to a residual moisture content of 5-10%. Store at room temperature.

The fermentation process in the laboratory carried out deep way in flasks with a capacity of 750 cm3on shaking the device with a speed of 160-180 min-1.

The main raw material for preparation of nutrient medium is sugar.

Preparing a nutrient medium for cultivation of seed spawn:

50.0 g of sugar, 0.16 g of dihydroorotate potassium, 2,50 g of ammonium nitrate, 0.25 grams magnesium sulfate of heptahydrate, 17 g of sugar beet molasses (to stimulate growth) was dissolved in 1 DM3the tap water. The nutrient medium is sterilized in an autoclave current�m ferry 15 min and at a gauge pressure of 0.05 MPa for 30 min, cooled to 32°C.

Culture of the fungus grown on wort-agar for 10 days, or air-dry conidia (107conidia/cm3medium) seeded with 50 cm3nutrient medium and grown mycelium in the seed for 36 h at a temperature of 32°C.

Sowing mycelium of 10 cm3seeded 50 cm3fermentation medium and the fermentation is carried out for 6 days at a temperature of 32°C. the Composition of the fermentation medium is different from the composition of the nutrient medium increased to 150 g/DM3sugar without the addition of molasses. During the fermentation process dolive nutrient medium not made. After the end of the cycle the culture of the fungus inactivated by boiling for 3 min and determine the amount of citric acid, the yield from sugar, eat citric acid per unit volume of medium per day of fermentation. The data presented in table 2.

EXAMPLE 2

Cooking the seed of a new strain of the nutrient medium for the cultivation of seed mycelium and the seed growing mycelium was carried out analogously to example 1.

Fermentation nutrient medium is carried out at a temperature of 32°C for 4.5 days.

The main raw material for the preparation of the fermentation medium is rye flour.

Preparation of suspensions of drug Cellularity":

1 g of the drug "Cellularity" with activity of 400 units. CMCS/g dissolved in 5 cm3tap water, incubated at 20°C for 30 min and the volume was adjusted to 50 cm3.

Preparation of suspensions of drug "Aminocoumarin":

1 g of the drug "Aminocoumarin" with the activity of 1000 units ░ C/g dissolved in 5 cm3tap water, incubated at 20°C for 30 min and the volume was adjusted to 50 cm3.

Preparation of nutrient medium for fermentation:

232 g of rye flour pour tap water at 20°C, the volume was adjusted to 500 cm3and incubated at 20°C for 16 h, then heated to 40°C, receiving of 44.2 wt.%-ing the suspension of rye flour (based on dry matter) content of carbon and nitrogen in a ratio of 14:1, and injected a suspension of enzyme preparation "Cellularity" in the amount of 2.5 cm3the content of karboksimetilcelljulozy 0.05 g, which corresponds to a dosage of 0.00025 grams per 1 g of raw material. The suspension with constant stirring, maintained at 40°C for 60 min, the drug is administered "Aminocoumarin" in the amount of 17.4 cm3the content of α-amylase 0,348 g, which corresponds to a dosage of the drug 0.0015 g per 1 g of raw material. With constant stirring, bring the temperature up to 85°C and maintained at this temperature For 60 min. terminating an enzyme preparation obtained hydrolysate was heated to 95°C, cooled to 2°C.

Then the rye flour hydrolysate was sterilized in the autoclave the current ferry 15 min and at pressure of 0.08 MPa for 30 min, cooled to 50°C and aseptically injected 10.0 cm3solution of ammonium nitrate concentration of 10 wt.%, 2.5 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.%, 0.8 cm3solution of potassium dihydrogen phosphate concentration of 10 wt.%, 0.5 cm3solution of sulphate of zinc heptahydrate concentration of 1.0 wt.% and the volume was adjusted to 1.0 DM3sterile water. The concentration of fermentable sugars 116,0 g/DM3.

The data presented in table 2.

EXAMPLE 3

Cooking the seed of a new strain, the culture medium for seed growing mycelium and seed growing mycelium was carried out analogously to example 1. Preparation of suspensions of enzyme preparations analogously to example 2.

The main raw material for the preparation of the fermentation medium is corn starch.

For fermentation broth prepare a suspension of 160 g of corn starch, getting to 28.5 wt.% suspension (calculated on dry substance), which is heated with vigorous stirring to 60°C and injected the enzyme preparation "Aminocoumarin" in the amount of 6.9 cm3the content of α-amylase was 0.138 g, which corresponds to a dosage of 0.001 g to 1 g of material. With constant stirring, bring the temperature� to 82°C and maintained at this temperature for 90 min, getting the hydrolysate with a DE=25%, providing glucose to 3.3%. For termination of an enzyme preparation obtained hydrolysate was heated to 95°C, then cooled to 20°C and diluted with sterile water to a concentration of fermentable sugars of 140 g/DM3.

In the resulting starch hydrolysate for fermentation is sterile injected with a solution of ammonium nitrate in the amount of 10.3 g/DM3concentration of 10 wt.%, providing a mass concentration of 2.5 g/DM3sterile autolysis of the mycelium of a culture of Aspergillus niger in an amount that provides a ratio of carbon and nitrogen 75:1, 2.5 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.%, providing the mass concentration of 0.25 g/DM3, 1.6 cm3solution of potassium dihydrogen phosphate concentration of 10 wt.%, providing the mass concentration of 0.16 g/DM3.

Fermentation nutrient medium is carried out at a temperature of 32°C for 5 days.

The data presented in table 2.

EXAMPLE 4

Cooking the seed of a new strain, the culture medium for seed growing mycelium and seed growing mycelium was carried out analogously to example 1. Preparation of suspensions of enzyme preparations analogously to example 2.

Preparation of nutrient medium for fermentation was carried out analogously to example 3, but prepare a suspension of pshenichnov� starch, getting hydrolysate with DE=30%.

Fermentation nutrient medium is carried out at a temperature of 32°C for 5 days.

The data presented in table 2.

EXAMPLE 5

Cooking the seed of a new strain, the culture medium for seed growing mycelium and seed growing mycelium was carried out analogously to example 1. Preparation of suspensions of enzyme preparations analogously to example 2.

Preparation of nutrient medium for fermentation was carried out analogously to example 3, but prepare a suspension of potato starch, receiving the hydrolysate with a DE=18%.

Fermentation nutrient medium is carried out at a temperature of 32°C for 5 days.

The data presented in table 2.

EXAMPLE 6

Cooking the seed of a new strain, the culture medium for the seed growing mycelium, the seed growing mycelium was carried out analogously to example 1.

Preparation of suspensions of enzyme preparations and the fermentation is carried out analogously to example 2.

The main raw material for the preparation of the fermentation medium is grinding wheat.

For fermentation broth prepare a suspension of 200 g of grinding wheat and 600 cm3tap water, getting 19 wt.%-ing the suspension of grinding wheat (based on dry matter) content of carbon and nitrogen in a ratio of 15:1. Enter 0.6 cm3 solution of ammonium nitrate concentration of 10 wt.%, 1.5 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.%, 0,48 cm3solution of potassium dihydrogen phosphate concentration of 10 wt%.

The concentration of fermentable sugars 120,0 g/DM3(PL.2).

The data presented in table 2 demonstrate that the new Cam system for true strain 02149 grown on Saharsa-the mining environment, significantly exceeds the known strain VKPM F-501 according to the intensity of biosynthesis of citric acid per unit volume per day and yield of citric acid from spent sugar. New strain allows the use of different types of starchy raw materials for the production of citric acid by fermenting a nutrient medium on the basis of hydrolysates of flour from rye grain, hydrolysates of grinding wheat, starch hydrolysates, and to achieve the intensity of biosynthesis of citric acid to 18.4-24.8 g/(DM3·day), the conversion of sugars into citric acid and 84.7-99.3 percent, while the strain VKPM F-501 these indicators on starchy raw material is much lower: the intensity of biosynthesis of citric acid and 17.1 g/(DM3·day), the conversion of sugars into citric acid is 77.2%.

Thus, the selected Cam system for true strain 02149 is versatile enough active kislotoobrazoutei and is available for fermentation, Saharsa-mineral media and environments, obtained as a result�ATA hydrolysis of starchy raw materials, providing high performance process.

Table 1
Comparison of cultural, morphological and physiological-biochemical characteristics of a new strain of Aspergillus niger Cam system for true 02149 with Aspergillus niger VKPM F-501
SignsIndicatorsThe name of the strainDifferences:
- no or low
+ there are
Cam system for true 02149VKPM F-501
12345
1. Cultural characteristics on wort-agar medium1.1 Painting of conidialight beigelight beige-
1.2 the colony Diameter, µm8194+
1.3 View of the reverse side of the colonysmooth, whiteradially �kletchataya, white+
2. Morphological features on wort-agar medium2.1 the Diameter of conidial heads, µm40-125100-140+
2.2 Length of sterigma of the first order, µm12,011,6-
2.3 Length of sterigma second-order7,34,5+
2.4 the diameter of the conidia, µm4,53,4+

Continuation of table 1
2. Morphological features on wort-agar medium2.5 Length of canadianese, µm510-1410270-1000+
2.6 the Diameter of canadianese, µm11-229-18-
3. Physiological and biochemical characteristics 3.1 Temperature range of growth,28-3630-34+
3.2 Optimum temperature, °C3232-
3.3 Optimum pH1,7-7,06,8-7,2+
3.4 sources of nitrogenAssimilates nitrogen organic compounds (peptone, protein, amino acids, yeast autolysis, mycelial mass, urea), assimilates the mineral nitrogen compounds, ammonium salts, nitratesAssimilates nitrogen organic compounds (protein, peptone, amino acids, yeast autolysis, urea), assimilates ammonium salts, nitrates-

Continuation of table 1
3. Physiological and biochemical characteristics3.5 Relation to carbon sourcesWell learns and grows on molasses, saccharose, glucose, starch hydrolysates, hydrolysates of flour from rye grain, rice and hydrolysates of cereal milling rye, wheat, ri�and, containing glucose, maltose, dextrinsUses glucose, fructose, sucrose, maltose, to a lesser extent beckons, galactose, sorbitol+
3.6 Conversion of sugars into citric acid, %84,7 being 99.382,2+
3.7 the Intensity of biosynthesis, g/(DM3·day)18,4-24,818,2+

Table 2
Indicators of the fermentation process
№ p/pName of raw materialsThe amount of sugar in the flask, gThe duration of fermentation, dThe name of the strainThe amount of citric acid, g/flaskThe content of citric acid in the amount of synthesized acid %Conversion of sugars to citric acid, %The intensity of biosynthesis of citric acid, g/(DM3·day)
1 23456789
1Saharsa-mineral Wednesday8,06,0Cam system for true 021497,2496,090,520,1
VKPM F-5016,5694,582,218,2
2Hydrolyzed rye flour DE=32%5,84,5Cam system for true 021494,9793,485,718,4
VKPM F-5014,6292,079,617,1
3Hydrolyzed corn starch DE=25%5,0Cam system for true 021497,4598,099,324,8
VKPM F-5016,9896,193,123,3

Continuation of table 2
4Hydrolysed potato starch DE=18%7,55,0Cam system for true 021496,9895,293,123,3
VKPM F-5016,4492,785,9A 21.5
5The hydrolysate of pre�ary starch DE=30% 7,55,0Cam system for true 021497,1596,295,323,8
VKPM F-5016,3094,884,021,0
6Hydrolysate grind wheat DE=35%6,04,5Cam system for true 021495.08 mm92,984,718,8
VKPM F-5014,6388,777,217,1

The strain of the fungus Aspergillus niger RCAM02149 - product of citric acid.



 

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The invention relates to the microbiological industry, and relates to a method of cleaning solutions of citric acid and related its biosynthesis kislotostabilen amylolytic enzymes with getstringarray and saharaganj activities

The invention relates to the microbiological industry, and relates to a method of obtaining from starch-containing raw materials citric acid and acid enzymes:-amylase and glucoamylase

FIELD: biotechnology.

SUBSTANCE: strain Aspergillus oryzae 12-84, having a high level of synthesis of the complex of proteases and peptidases, nucleases, chitinase, β-glucanase, mannanase and α-amylase, is deposited in State Scientific Institution of All-Russian Scientific Research Institute of Agricultural Microbiology of the Russian Agricultural Academy under the registration number Aspergillus oryzae RCAM01134. The strain may be used to produce complex enzyme preparations with their subsequent application for hydrolysis of raw materials in the preparation of biocorrectors of food and feed, amino acid additives and biologically active additives with functional properties.

EFFECT: invention enables to increase the yield of proteolytic, nuclease, chitinase, β-glucanase, mannanase and α-amylase activities.

2 tbl, 2 ex

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