The method of obtaining citric acid

 

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 method involves the hydrolysis of starch by enzyme drug bacterial-amylase at elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom-kislotoobrazoutei Aspergillus niger. In a nutrient medium for fermentation additionally add mineral salts in the form of sulphate of copper (II), zinc, cobalt (II), respectively (0,5-1,0)10-3, (1,5-3,5)10-3, (1,5-4,5)10-3g 1 DM3hydrolyzed starch and organic nitrogen source in an amount to provide content in the environment of carbon and nitrogen in the ratio (65-75):1, respectively. The result of the invention is the conversion of sugars to citric acid level (79,5-83,2% and getting acid enzymes:-amylase and glucoamylase with activities accordingly 0,80-1,25 units A./cm3and 32.6-76,7 unit is STI and concerns a method for obtaining from starch-containing raw materials citric acid and acid enzymes:-amylase and glucoamylase.

A method of obtaining amylolytic enzymes cultivation of the fungus Aspergillus niger at different starch-containing media with the release of 0.64 units/cm3-amylase and 15.7 u/cm3glucoamylase /1/.

In this way receive only acid enzymes-amylase and glucoamylase.

The closest present invention is a method for citric acid-based medium, containing hydrolyzed starch and mineral salts, including the hydrolysis of a starch suspension containing 26-36 wt. % of dry substances, enzyme drug bacterial-amylase, taken in an amount of 0.5-1.5 units amylolytic capacity per 1 g of dry substance of the starch, while keeping at a pressure of 0.1 MPa for 30 minutes, the addition of mineral salts to the hydrolyzed starch with a value dextrose equivalent value not exceeding 30, and subsequent fermentation of a nutrient medium mushroom - kislotoobrazoutei Aspergillus niger for 6 days at a temperature of 32oC. After fermentation the culture of the fungus inactivate boiling and determine the conversion of sugars to citric acid /2/.

mylasa are formed in small quantity. Information about the simultaneous receipt of citric acid and acid amylolytic enzymes are missing.

The technical result of the invention is to obtain the target products of microbiological synthesis of citric acid three products: citric acid, acid amylolytic enzymes-amylase and glucoamylase, increasing their output.

The technical result of the invention is achieved in that in a method of producing citric acid, including the hydrolysis of starch by enzyme drug bacterial-amylase at elevated temperature and pressure to achieve value dextrose equivalent of hydrolyzed starch not more than 30, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom - kislotoobrazoutei Aspergillus niger add additional mineral salts in the form of sulphate of copper (II), zinc, cobalt (II), respectively (0,5-1,0)10-3, (1,5-3,5)10-3, (1,5-4,5)10-3g 1 DM3hydrolyzed starch and organic source of nitrogen in Col, confirming the possibility of achieving a technical result of the invention, represented in the examples.

As a source of starch-containing raw material using a starch slurry with a concentration of dry substances of 28.5 wt.%, made from dry starch content of 87 wt. % of dry substances, as well as bacterial enzyme preparation-amylase using a product manufactured by the domestic industry under the trade name "Aminocoumarin GSH" or "Aminocoumarin GH" activity from 1000 to 2500 units amylolytic ability (A.) on 1 g of the drug, the drug use 1000 A. With/g as a producer of target products used strains of the fungus Aspergillus niger VKPM F-171, VKPM F-501 and VKPM F-719 known producers of citric acid in bioconversion beet molasses /3, 4/ and sorghum juice /5/.

As the organic nitrogen source used hydrolyzed soy flour, hydrolyzed corn flour, autolysate mycelium of the fungus Aspergillus niger.

Preparation of hydrolyzed soy flour concentration of 100 g/DM3. A portion of 100 g of soya flour suspended in 700,0 cm3the tap water. To a suspension add drug Prothalamion HH the hours The volume of the obtained hydrolysate was adjusted with water to 1000 cm3and maintained at a gauge pressure of 0.1 MPa for 30 min, then cooled to 45-50oC. Hydrolyzed soy flour concentration of 100 g/DM3contains of 7.36 % nitrogen /6/.

Preparation of hydrolyzed corn flour concentration of 500 g/DM3. The portion 500 g corn flour suspended in 700,0 cm3the tap water. To a suspension add drug Aminocoumarin GSH or GH at the rate of 1.5 units per 1 g of corn flour and carry out the enzymatic hydrolysis at a temperature of 80-85oC for 1.5 h, the Volume of the obtained hydrolysate was adjusted with water to 1000 cm3and maintained at a gauge pressure of 0.1 MPa for 30 min, then cooled to 45-50oC. the Hydrolysate of corn flour a concentration of 500 g/DM3contains 1.2 % nitrogen /6/.

Preparation of autolysate mycelium of the fungus Aspergillus niger concentration of 80 g/DM3. The hinge 80 g of wet mycelium (humidity 80%) are suspended in tap water, heated to 50-52oC. the Volume was adjusted with water to 1000 cm3, is maintained at a gauge pressure of 0.1 MPa for 60 min, then cooled to 45-50oC. Autolysate mycelium of the fungus Aspergillus niger concentration of 80 g/DM3contains 8% nitrogen. Nitrogen has been determined using the

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 32oC.

b) Preparation of culture medium for seed growing mycelium.

11.5g starch dissolved in a preheated 50oWith tap water to bring the volume to 200 cm3getting 5,12 wt.% starch suspension (calculated on dry substance starch).

With vigorous stirring, heat the starch suspension to 55-60oWith and enter 2 wt.%-s 'solution of enzyme preparation bacterialamylase in an amount to provide liquefaction of starch, respectively dextrose equivalent (DE) equal to 25.0, with constant stirring, and incubated for 90 min To stop the enzyme action of the drug hydrolyzate is heated to boiling, cooled to 20-22oAnd the volume was adjusted to 200 cm3.

Then hydrolyzed starch with a DE, equal to 25.0, vyderzhivatel ammonium nitrate concentration of 10 wt.%, 0,50 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.% and 0.30 cm3solution of potassium dihydrophosphate concentration of 10 wt.%.

C) Preparation of culture medium for fermentation 160,0 g of starch dissolved in water heated to 50oC, the volume was adjusted to 500 cm3getting to 28.5 wt.%-percent starch suspension (calculated on dry substance starch), which is heated with vigorous stirring to 55-60oWith and enter an enzyme bacterial-amylase in an amount to provide DE, equal to 25.0. With constant stirring, bring the temperature up to 82-85oC and maintained at this temperature for 90 minutes To stop the enzyme action of the drug hydrolyzate is heated to boiling, cooled to 20-22oC, the volume was adjusted to 500 cm3.

Then the hydrolysate is maintained at a gauge pressure of 0.1 MPa for 30 min, cooled to 45-50oWith and sterile contribute to 18.9 cm3solution of ammonium nitrate concentration of 10 wt.%.

As an organic source of nitrogen contribute sterile hydrolyzed soy flour concentration of 100 g/DM3the number 26,9 cm3providing in the ratio of carbon and nitrogen as 65:1. Then in a solution of potassium dihydrophosphate concentration of 10 wt.%, 0.5 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.%, which corresponds to 0.0025 g of salt, 0.3 cm3solution of cobalt sulfate heptahydrate concentration of 0.5 wt. %, which corresponds to 0.0015 g cobalt salts, 0,20 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%, which corresponds 0,0010 g of salt of copper, and diluted with sterile water to 1.0 DM3. Concentration formatiruem sugars - (150-160 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 32oC.

d) Fermentation of a nutrient medium on the basis of hydrolyzed starch citric acid In a flask with a capacity of 750 cm3placed 50 cm3the nutrient medium and seeded with its 10 cm3rising mycelium. The flask was placed on a rocking chair with a speed of 160 min-1and incubated for 6 days at a temperature of 32oC. 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 fermentati (CH.C).

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 using hydrolyzed starch with a DE, equal 26,5.

In the hydrolyzed starch enter solution of ammonium nitrate concentration of 10 wt. % number 17,6 cm3and sterile hydrolyzed corn flour concentration of 500 g/DM3the number 30,1 cm3providing in the ratio of carbon and nitrogen as 70:1, 0.3 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.%, appropriate 0.0015 g zinc salts, 0.9 cm3solution of cobalt sulfate heptahydrate concentration of 0.5 wt. % corresponding 0,0045 g of salt of cobalt, 0.10 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%, appropriate 0.0005 g of salt of copper.

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 the cultivation of pseudotolithus starch with a DE, equal to 28.5, and hydrolyzed starch for fermentation enter solution of ammonium nitrate concentration of 10 wt. % in the amount of 16.4 cm3sterile suspension of autolysate mycelium culture of Aspergillus niger concentration of 80 g/DM3in number to 66.4 cm3providing in the ratio of carbon and nitrogen as 75:1. Then make sterile 0.7 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.%, which corresponds 0,0035 g zinc salts, 0.3 cm3solution of cobalt sulfate heptahydrate concentration of 0.5 wt.%, appropriate 0.0015 g cobalt salts, 0,20 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%, appropriate 0,0010 g of salt of copper.

Example 4.

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, but using hydrolyzed starch with a DE, equal 29,0, it make sterile solution of ammonium nitrate concentration of 10 wt.% in the amount of 18.9 cm3as the organic nitrogen source hydrolyzed soy flour concentration of 100 g/DM3in the amount of 17.7 cm3suspension autolysate mycelium of the fungus Aspergillus niger concentration of 80 g/DM350 cm3that obesptechit a concentration of 0.5 wt.%, appropriate 0,002 grams zinc salts, 0.5 cm3solution of cobalt sulfate heptahydrate concentration of 0.5 wt.%, appropriate 0.0025 g of salt of cobalt and 0.2 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%, appropriate 0,0010 g of salt of copper.

Example 5 (the prototype).

Preparation of conidia producer - fungus Aspergillus niger strain VKPM F-501, seed growing mycelium fermentation of a nutrient medium for fermentation is conducted analogously to example 1, but without addition of sulphate of zinc, cobalt, copper and organic source of nitrogen.

The technical result in the experiments presented in examples 1-4 of table, in comparison with the prototype is to maintain the conversion of sugars to citric acid at a sufficiently high level (79,5-83,2% and getting acid enzymes:-amylase and glucoamylase with activities increased accordingly to 0.80-1.25 units A./cm3and 32.6-76,7 units CH. With a/cm3.

Sources of information 1. Tokhadze H. C., Kvachadze L. P., Kvesitadze, I. Influence of nutrient medium composition on acid biosynthesis-amylase different species of Aspergillus// Applied biochemistry and Microbiology. - 1975 - 4 - n-515-518.

2. RF patent 2132384, Mr> 5. RF patent 2088658, IPC 6 12 N 1/14, 12 P 7/48, 1997.

6. The chemical composition of food. Reference book/ Ed. by Acad. The Academy of medical Sciences of the USSR A. A. Pokrovsky - M: Food industry - 1977 - n-18.24.

Claims

The method of obtaining citric acid, including the hydrolysis of starch by enzyme drug bacterial-amylase at elevated temperature and pressure to achieve value dextrose equivalent of hydrolyzed starch not more than 30, the addition of mineral salts to the hydrolyzed starch and the subsequent fermentation medium mushroom-kislotoobrazoutei Aspergillus niger, characterized in that add additional mineral salts in the form of sulphate of copper (II), zinc, cobalt (II), respectively (0,5-1,0)10-3, (1,5-3,5)10-3, (1,5-4,5)10-3g 1 DM3hydrolyzed starch and organic nitrogen source in an amount to provide content in the environment of carbon and nitrogen in the ratio (65-75):1, respectively.

 

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