Method for preparation of citric acid, ?-amilase and glucoamylase

FIELD: chemistry.

SUBSTANCE: method provides the hydrolysis of starch-containing raw materials, following adding of the mineral salts to the obtained hydrolysate and medium fermentation with acid-forming fungi Aspergillus niger. The hydrolysis is carried out with ferment preparate of bacterial α-amilase at increased temperature and excessive pressure. The starch-containing raw material are rice flour allowed beforehand in the water at 20-25°C during 10-12 hrs. in amount of 205.3-235.7 g and rye flour in amount of 232.0-290.6 g. The carbon and nitrogen ratio in the obtained rice flour suspension is provided respectively equal (20.0-23.0):1, in rye flour suspension - (14.0-16.0):1. The ferment preparate is added up to achieving of carbon source dextrose equivalent value 35-40%. The sugars conversion to citric acid is 86.7-93.2%. α-amilase and glucoamylase activity is respectively 4.5-6.2 units A,C/cm3 and 160-180 units Gl.C/cm3.

EFFECT: increase of α-amilase and glucoamylase yield while maintaining the high yield of citric acid.

1 tbl, 7 ex

 

The invention relates to the microbiological industry, and relates to a method of obtaining from starch-containing raw materials citric acid and acid enzymes: α-amylase and glucoamylase, which is used in baking, brewing, starch industry, medicine, i.e. all processes requiring hydrolysis of poly - and oligosaccharides.

A method of obtaining citric acid, including the hydrolysis of starch by drug bacterial α-amylase at elevated temperature and pressure to achieve value dextrose equivalent of hydrolyzed starch not less than 30%, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom-kislotoobrazoutei Aspergillus niger. As a mineral salt use salt in the form of sulphate of copper (II), zinc, cobalt (II). The hydrolyzed starch add additional organic nitrogen source in an amount to provide content in the environment of carbon and nitrogen in the ratio (65-75):1 /1/.

The disadvantage of the above method is the low output OC-amylase and glucoamylase.

Closest to the proposed method is a method of obtaining citric acid, α-amylase and glucoamylase, including the hydrolysis of starch-containing raw materials of the enzymatic preparation of bacterial α-amylase is ri elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom-kislotoobrazoutei Aspergillus niger /2/.

The disadvantage of the above method is the low yield of α-amylase and glucoamylase.

The technical result of the invention is to increase the yield of α-amylase and glucoamylase while maintaining a high yield of citric acid.

The technical result of the invention is achieved in that in a method of producing citric acid, α-amylase and glucoamylase, including the hydrolysis of starch-containing raw materials of the enzymatic preparation of bacterial α-amylase at elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom - kislotoobrazoutei Aspergillus niger, as a starch-containing raw materials use rice in the number 205,3-KZT 235.7 g or rye flour in the number 232,0-290,6 g pre-aged in water at 20-25°C for 10-12 hours, the content in the resulting suspension of rice flour carbon and nitrogen in the ratio (20-23):1, and in the suspension of rye flour (14-16,0):1, respectively, the enzyme preparation used in the number to reach the value dextrose equivalent source of carbon is not less than (35-45)%.

Use as feedstock rice m the Ki in the number 205,3-KZT 235.7 g or rye flour 232,0-290,6 g, as well as the prior artificial rice or rye flour in water at 20-25°C for 10-12 hours helps maximum output from the plant cells of soluble carbohydrates and protein needed for growth and development of the fungus Aspergillus niger, which provides the ratio of carbon and nitrogen (20-23):1 slurry of rice flour and (14-16):1 - for the suspension of rye flour, reduce the duration of hydrolysis and increases the yield of α-amylase and glucoamylase.

Use of the enzyme preparation in an amount up to reach the value dextrose equivalent carbon source 35-45% also increases the yield of α-amylase and glucoamylase, while maintaining high yield of citric acid.

As a source of starch-containing raw material using a suspension of rice or rye flour with a concentration of solids of 25 wt.%, made with dry rice or rye flour with a solids content of 86 wt.%, and as the enzyme preparation of bacterial α-amylase using a product manufactured by the domestic industry under the trade name "Aminocoumarin GSH" or "Aminocoumarin GH" activity from 1000 to 3000 units amylolytic ability (A.S.) on 1 g of the drug, the drug use 1000 AS/g, and as a producer of target products selected strains of the fungus Aspergillus niger VKPM F-171, VKPM F-501 and VKPM F-719, zvezdnye producers of citric acid for the bioconversion of sugar beet molasses /2, 3/ and sorghum juice /4/.

The method is illustrated by the following examples.

Example 1

a) Preparation of conidia producer

Conidia of the fungus-producer Aspergillus niger strain VKPM F-171 are weighed into a sterile tube in the calculation of 250 mg / 100 cm3, placed in a medium of the following composition, g/DM3: sugar - 50,0, dihydroorotase potassium - 0,16, ammonium nitrate - 2,50, magnesium sulfate heptahydrate - 0,25.

A suspension of conidia of the fungus in the flask is placed on the rocking chair with the speed of 160 min-1and incubated for 5-6 hours at a temperature of 32°C.

b) Preparation of nutrient media for cultivation of seed mycelium

50.0 g of sugar, 0.16 g of dihydroorotate potassium, 2.50 g of ammonium nitrate, 0.25 g of magnesium sulfate heptahydrate dissolved in 1 DM3the tap water. Then the solution is maintained at a gauge pressure of 0.1 MPa for 30 min, cooled to 25-32°C.

C) Preparation of culture medium for fermentation

205,3 g of rice flour filled with tap water at a temperature of 20°C, the volume was adjusted to 500 cm3and maintained at a temperature of 20°C for 10 hours, then heated to 40°C receives 41,0 wt.%-ing the suspension of rice flour (calculated on the dry matter) content of carbon and nitrogen in the ratio 23:1, which is heated with vigorous stirring to 55-60°C and injected an enzyme of bacterial α-amylase in the share of the ve, providing DE, equal to 35%. With constant stirring, bring the temperature up to 82-85°C and maintained at this temperature for 60 minutes For the termination of enzyme preparation obtained hydrolyzate is heated to boiling, cooled to 20-22°C, the volume was adjusted to 500 cm3.

Then hydrolyzed rice flour with DE, equal to 35%, is maintained at a gauge pressure of 0.1 MPa for 30 min, cooled to 45-50°C and sterile contribute 0.5 cm3solution of ammonium nitrate concentration of 10 wt.%, 1.25 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.%, 0,80 cm3solution of potassium dihydrophosphate concentration of 10 wt.%, 0.5 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.% and diluted with sterile water to 1.00 DM3. Concentration formatiruem sugars to 130.6 g/DM3environment.

g) seed Growing mycelium

In a flask with a capacity of 750 cm3placed 50 cm3the nutrient medium and seeded on 10 cm3suspensions of conidia. The flask was placed on a rocking chair with a speed of 160 min-1and incubated for 36 hours at a temperature of 32°C.

d) Fermentation of a nutrient medium on the basis of hydrolyzed rice flour

In a flask with a capacity of 750 cm3placed 50 cm3the nutrient medium and seeded her 9 cm3rising mycelium. The flask was placed on a rocking chair with a speed of 160 min-1and provide the claim within 5 days at a temperature of 32°C. After fermentation the biomass of the fungus is separated in a Buechner funnel and in the culture solution determine the conversion of sugars to citric acid and the acid activity of amylolytic enzymes: α-amylase, - amylase ability, glucoamylase - glucoamylase ability (GLS).

Example 2

a) Preparation of conidia producer - fungus Aspergillus niger strain VKPM F-501, seed growing mycelium fermentation of a nutrient medium is conducted analogously to example 1.

b) Preparation of nutrient media for growing seeds and mycelium preparation of culture medium for fermentation is conducted analogously to example 1, but the suspension of rice flour soak in tap water for 12 hours at a temperature of 25°C, the hydrolysis get DE equal to 40%.

Example 3

a) Preparation of conidia producer - fungus Aspergillus niger strain VKPM F-719, seed growing mycelium fermentation of a nutrient medium is conducted analogously to example 1.

b) Preparation of nutrient media for growing seeds and mycelium preparation of culture medium for fermentation is conducted analogously to example 1, the aging time in the water 11 hours, and for the preparation of the hydrolysate used KZT 235.7 g of rice flour, receiving of 47.1 wt.%-ing the suspension of rice flour (calculated on the dry matter) content of carbon and nitrogen in sootnoshenie is to 20.0:1, and use hydrolyzed rice flour with DE, equal to 45%. Concentration formatiruem sugars 150,0 g/DM3environment.

Example 4

Preparation of conidia producer - fungus Aspergillus niger strain VKPM F-171, seed growing mycelium fermentation of a nutrient medium is conducted analogously to example 1, but preparing a suspension of 232,0 g rye flour, getting to 46.4 wt.%-ing the suspension of rye flour (calculated on the dry matter) content of carbon and nitrogen in the ratio of 14:1, and use the hydrolysate with a DE, equal to 40%. Concentration formatiruem sugars 119,7 g/DM3environment.

Example 5

Use the producer - fungus Aspergillus niger strain VKPM F-501, preparation of conidia and the fermentation is carried out analogously to example 1, but preparing a suspension of 271,3 g rye flour, incubated at 25°C for 11 hours, getting to 54.2 wt.%-ing the suspension of rye flour (calculated on the dry matter), get the suspension rye flour content of carbon and nitrogen in the ratio of 15.4:1.0 and use the hydrolysate with a DE, equal to 37%. Concentration formatiruem sugars 140,9 g/DM3environment.

Example 6

Use the producer - fungus Aspergillus niger strain VKPM F-719, preparation of conidia and the fermentation is carried out analogously to example 1, but prepare to 58.1 wt.%-ing the suspension of 290,6 g rye flour, which is kept in tap water for 12 hours at a temperature of 22°C receives susp is SIU with carbon and nitrogen in the ratio of 16:1, use a hydrolysate with a DE, equal to 43%. Concentration formatiruem sugars 149,9 g/DM3environment.

Example 7 (the prototype).

Preparation of conidia producer - fungus Aspergillus niger strain VKPM F-501, seed growing mycelium, preparation of culture medium for fermentation is conducted analogously to example 1, but used for preparation of the nutrient medium, the suspension of 140.0 g of starch and 20 g of corn flour heated to 40°C. tap water to bring the volume to 500 cm3and heated to 40-50°C receives 32,0 wt.%-ing the suspension, drug bacterial α-amylase in an amount to provide DE, equal to 26%, making 25.5 cm3solution of ammonium nitrate concentration of 10 wt.%, providing in the ratio of carbon and nitrogen 50:1. Concentration formatiruem sugars 135,4 g/DM environment.

The technical result in the experiments presented in examples 1-6 table, compared with the prototype is to increase the conversion of sugars to citric acid to (86,7-93,2% and increasing the activity of enzymes: α-amylase and glucoamylase respectively to 4.5 to 6.2 units AS/cm3and 160-180 units PS/cm3.

SOURCES of INFORMATION

1. RF patent №2215036, publ. 2003

2. RF patent №2266960, publ. 2005

1. RF patent №975799, publ. 1982.

2. Patent USSR No. 1811697, publ. 1993.

3. RF patent №208658, publ. 1997.

The method of obtaining citric acid, α-amylase and glucoamylase, including the hydrolysis of starch-containing raw materials of the enzymatic preparation of bacterial α-amylase at elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom-kislotoobrazoutei Aspergillus niger, characterized in that as the starch-containing raw materials use rice in the number 205,3-KZT 235.7 g or rye flour in the number 232,0-290,6 g pre-aged in water at 20-25°C for 10-12 h, providing content in the resulting suspension of rice flour of carbon and nitrogen in the ratio (20,0-23,0):1, and in the suspension of rye flour (14,0-16,0):1, respectively, the enzyme preparation used in the number to reach the value dextrose equivalent carbon source 35-40%.



 

Same patents:

FIELD: microbiological industry.

SUBSTANCE: citric acid is obtained by fermentation of fungi Aspergillus niger. Fermentation is carried out in periodic mode in at least three fermenters wherein one of these fermenters is the head one, to produce in head fermenter organic acid concentration of 110-140 g/dm3 and sugar concentration of 3-5 g/dm3. and in the rest fermenters 80-90 g/dm3 and 10-12 g/dm3, respectively. Fermentation id continuous mode is carried out in two step in fermenter battery connected in parallel with the head on, wherein organic acid and sugar concentrations are maintained at 12-16 g/dm3, and viscosity is maintained at 0.04-0.05 Pa.s. In this purpose cultural liquid with sugar concentration of 3-5 g/dm3 is periodically evacuated from head fermenter and cultural liquid with sugar concentration of 10-12 g/dm3 is evacuated from the rest ones, and nutrient solution and sterile water are added thereto.. Additionally carbohydrate sources and mineral salts are introduced.

EFFECT: increased citric acid yield due to increased fermenter productivity.

2 cl, 1 tbl, 3 ex

FIELD: microbiological industry, biochemistry.

SUBSTANCE: invention relates to a method for preparing citric acid and a complex of acid-stable amylolytic enzymes possessing activity of α-amylase and glucoamylase. Method involves separation of mycelium of fungus Aspergillus niger as a producer of acid from a fermented solution with the protein concentration 1.5-7.0 g/dm3 at temperature ≤32°C. Prepared fermented solution is purified by successive filtration processes through membranes with pores diameter values 0.65, 0.45 and 0.15 mcm and separated by ultrafiltration through a membrane retaining substances with molecular mass in limits 1000-50000 Da and providing preparing citric acid solution and acid-stable amylolytic enzymes solution. Citric acid solution with the protein content 0.05-0.15 g/dm3 is cleared preliminary followed by its concentrating, crystallizing and citric acid crystals are separated from mother liquor. Mother liquor with optical density value 0.1-0.4 at λmax = 400 nm is recovered to the concentrating step and solution with optical density 0.41-0.8 at λmax = 400 nm is cleared preliminary and recovered to the concentrating step. Solution of above mentioned enzymes is dried by sublimation or spraying up to preparing a dry complex with enzymatic activity of α-amylase and glucoamylase 700-900 and 10000-15000 U/g, respectively, or solution is precipitated followed by drying up to preparing the dry complex with enzymatic activity of α-amylase and glucoamylase 1000-1300 and 20000-26000 U/g, respectively. Method provides simultaneous preparing crystalline citric acid and a dry complex of acid-stable amylolytic enzymes with the high quality and high yield.

EFFECT: improved preparing method.

2 tbl, 5 ex

FIELD: microbiological industry, in particular production of citric acid and acid-resistant α-amylase and glucoamylase enzymes.

SUBSTANCE: claimed method includes starch and maize flour hydrolysis in ratio of 1:7-1:40 with bacterial α-amylase enzyme preparation at elevated temperature and pressure to obtain value of carbon source dextrose equivalent not more than 30; addition of nitrogen organic source and mineral salts such as copper (II), zinc, cobalt (II) sulfates in amount of (0.5-1.0)x10-3, (1.5-3.5)x10-3, (1.5-4.5)x10-3 g/dm3, respectively, iron (II) to starch and maize flour hydrolyzate followed by medium fermentation with acid-forming fungi Aspergillus niger. Than hydrolyzate is diluted with sterile water to obtain fermentable sugar content of 120-140 g/dm3 and carbon and nitrogen organic source in ratio of (50-60):1, respectively. Method of present invention males in possible to convert sugar to citric acid with yield of 79.3-83.1 % and to obtain acid-resistant α-amylase and glucoamylase enzymes having activities of 2.50-3.50 U A-C/cm3 and 80.2-147.4 U Gl-C/cm3, respectively.

EFFECT: method for production of citric acid, α-amylase and glucoamylase with increased yield.

1 tbl, 4 ex

FIELD: biotechnology, microbiology, organic chemistry.

SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

EFFECT: improved preparing method.

1 tbl, 6 ex

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

The invention relates to the microbiological industry, in particular to the selection of the active strains - producers of citric acid

The invention relates to biotechnology, namely the method of separation of citric acid from solutions of alkali citrates

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, 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

FIELD: biotechnology, biochemistry.

SUBSTANCE: the strain Aspergillus awamori VKM F-3771 D is obtained by the multiple-stage genetic selection of the parent strain Aspergillus awamori VUD T-2 F-203 using ultraviolet irradiation. The strain elicits high activity of enzyme glucoamylase. For providing the high productivity of the strain nutrient media can be used that are used usually in industrial technologies in preparing similar enzyme preparations being without using complex procedures in culturing the strain-producer. Invention can be used for saccharification of starchy raw in different branches of food industry wherein highly active enzyme preparations with resistance to acid pH values are required.

EFFECT: valuable properties of strain.

1 tbl, 4 ex

The invention relates to biotechnology and can be used in the food industry with the aim of obtaining glucose during hydrolysis of starch

The invention relates to the microbiological industry, and can be used in the production of enzymes, protein, ethanol, animal feed, etc
The invention relates to biotechnology, can be used for saccharification of starchy raw materials in various food industries that require highly active enzyme preparations that are resistant to acidic pH values
The invention relates to biotechnology and can be used in the food industry with the aim of obtaining glucose during hydrolysis of starch

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-4150 obtained by selection from the known strain Aspergillus oryzae-387 (VKPM F-683) by multistep selection using effective methods of mutagenesis. The strain is stored as lyophilic dried culture and on slants with wort-agar in section of biotechnology of enzyme preparation, in food processing industry department of the State Scientific Institute of VNII food processing technology in Moscow. Invention provides preparing acid α-amylase showing high activity that exceeds activity in analogue by 2-3 times and reducing the process culturing time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

FIELD: biotechnology, microbiology, organic chemistry.

SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

EFFECT: improved preparing method.

1 tbl, 6 ex

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

The invention relates to the medical industry, in particular the microbiological synthesis of enzymes for medical purposes

FIELD: biotechnology, microbiology, organic chemistry.

SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

EFFECT: improved preparing method.

1 tbl, 6 ex

Up!