The method of obtaining citric acid

 

(57) Abstract:

The method of obtaining citric acid involves the hydrolysis of a starch suspension containing 26-30 wt.% dry substances, enzyme drug bacterial-amylase, taken in an amount of 1.5-2.0 units amylolytic ability (A. C.) for 1 kg of dry substance of the starch, at elevated temperature and pressure, adding to the hydrolyzed starch mineral salts in the form of sulphate of zinc, iron (II), copper in number (2,0-7,0)10-3g/DM3hydrolyzed starch, subsequent fermentation of a nutrient medium with the fungus Aspergillus niger. After fermentation and separation of the biomass of fungus get a culture solution containing 84,9 and 88.5 wt.% citric acid and acid enzymes: amylase and glucoamylase. The method allows to obtain a culture solution, which has increased amylolytic activity of acid enzymes: amylase and glucoamylase respectively, a unit of A. C. /cm3: of 0.62 to 0.69 and 22.5-28,6. table 1.

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.

Known SPO is the Reda with the release of 0.64 units/cm3-amylase and 15.7%/cm3glucoamylase [1].

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

Closest to the proposed 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 min, the addition of mineral salts of nutrients and subsequent fermentation of a nutrient medium mushroom - kislotoobrazoutei Aspergillus niger for 6 days at a temperature of 32oC. After fermentation the biomass inactivate boiling, separating the culture solution and define it as a conversion of sugars to citric acid [2].

In this way receive citric acid, and it can be assumed that a-amylase and glucoamylase are formed in small quantity. Information about the simultaneous receipt of citric acid and acid amylolytic enzymes are missing.

lot and additionally acid amylolytic enzymes: amylase and glucoamylase.

The technical result of the invention is achieved by a method of obtaining citric acid, comprising hydrolysis of a starch suspension containing 26-30 wt. % of dry substances, enzyme drug bacterial-amylase at elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch, subsequent fermentation of a nutrient medium mushroom - kislotoobrazoutei Aspergillus niger at a temperature not exceeding 32oC and the Department of culture solution in which according to the invention using bacterial-amylase, taken in an amount of 1.5-2.0 units amylolytic capacity per 1 g of dry substance of the starch, is added to the hydrolyzed starch addition of mineral salts in the form of sulphate of zinc, iron (II), copper in number (2,0-7,0)10-3g/DM3use the mushroom - kislotoobrazoutei Aspergillus niger strain VKPM F-171 and after fermentation is separated acidic culture solution having amylolytic and glucoamylase activity.

Information confirming the possibility of achieving a technical result of the invention, represented in the examples.

As a source of starch-containing raw materials Sparganium 88 wt.% dry matter, and as an enzyme preparation bacterial-amylase using a product manufactured by the domestic industry under the trade name "Aminocoumarin GH" activity from 1000 to 2500 units amylolytic ability (A. C.) on 1 g of the drug in the examples used the product with the activity of-amylase 2000 units A. S. /g, as a producer of target products use a known strain of the fungus Aspergillus niger, which is still used for the bioconversion of sugar beet molasses in citric acid (strain VKPM F-171) [3].

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

Potassium dihydrophosphate - 0,16

Ammonium nitrate - 2,5

Magnesium sulfate heptahydrate - 0,25

A suspension of conidia 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 nutrient media for cultivation of seed mycelium

11.9 g of dry starch dissolved in a preheated 50oWith Vodop the government of starch).

With vigorous stirring, heat the starch suspension to 58-60oWith and enter 2 wt.%-s ' solution of enzyme preparation of bacterial amylase in the number of 0.39 cm3that matches of 0.075 wt.% of the drug to the mass of dry substance starch or 1.5 units A. S./g of dry substance of the starch, with constant stirring, bring the temperature up to 82-85oC. For termination of enzyme preparation hydrolyzate is heated to boiling, cooled to 20-22oAnd the volume was adjusted to 200 cm3.

Then the hydrolyzed starch is maintained at a gauge pressure of 0.1 MPa for 30 min, cooled to 45-50oWith and sterile contribute 5 cm3solution of ammonium nitrate concentration of 10 wt.%, 0.5 cm3solution of sulphate of magnesium heptahydrate concentration of 10 wt. % and 0.32 cm3solution of potassium dihydrophosphate concentration of 10 wt.%.

C) Preparation of culture medium for fermentation

150 g of dry starch dissolved in water heated to 50oC, the volume was adjusted to 500 cm3getting to 26.4 wt.%-percent starch suspension (calculated on dry substance starch), which is heated with vigorous stirring to 58-60oWith and enter 2 wt.%-hydrated enzyme solution is wow substance of the starch, which corresponds to 1.5% of the A. S./g of dry substance of the starch, 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 12.5 cm3solution of ammonium nitrate concentration of 10 wt.%, 1.25 cm3solution of magnesium sulfate heptahydrate concentration of 10 wt.%, 0.8 cm3solution of potassium dihydrophosphate concentration of 10 wt. %, 0.5 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt. %, 0.2 cm3solution of sulphate heptahydrate iron concentration of 0.5 wt. %, 0.7 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.% and diluted with sterile water to a concentration of formatiruem sugars 150-155 g/DM3.

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 48 hours at a temperature of
In a flask with a capacity of 750 cm3placed 50 cm3the nutrient medium and seeded with its 10 cm3rising mycelium. The flask is placed on the rocking chair with the 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 activity of the acid enzymes and conversion of sugars to citric acid.

Example 2

a) Preparation of conidia producer Aspergillus niger 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 increase the dose of enzyme preparation bacterial-amylase to 2.0 units A. S./g of dry starch in the culture medium for fermentation is injected 0.2 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.%, 0.7 cm3solution of sulphate heptahydrate iron concentration of 0.5 wt.%, 0.5 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%.

Example 3

Preparation of conidia producer Aspergillus niger VKPM F-171, growing seed mitigatable nutrient medium for fermentation is conducted analogously to example 2, but in a nutrient medium for fermentation, made from 170,8 g of dry starch in the form of a 30 wt.% starch suspension, impose 0.7 cm3solution of sulphate of zinc heptahydrate concentration of 0.5 wt.%, 0.4 cm3solution of sulphate heptahydrate iron concentration of 0.5 wt.%, 0.2 cm3solution of copper sulfate pentahydrate concentration of 0.5 wt.%.

Example 4

Preparation of conidia producer of the fungus Aspergillus niger strain VKPM F-171, seed growing mycelium, preparation of nutrient media for cultivation of seed mycelium, preparation of culture medium for fermentation is conducted analogously to example 1, but without addition of sulphate of zinc, iron, copper. After fermentation, as in example 1, the biomass of the fungus is separated and in the culture solution determine the acid activity of amylolytic enzymes and conversion of sugars to citric acid.

Example 5 (prototype)

Preparation of conidia producer of the fungus Aspergillus niger strain VKPM F-171, preparation of nutrient media for cultivation of seed mycelium, preparation of culture medium for fermentation, seed growing mycelium fermentation of a nutrient medium on the basis of hydrolyzed starch citric acid spend Ana is the target. Then define the quality indicators of the culture solution.

These examples are presented in the table.

The comparison presented in table results of examples 1-4 (according to the invention) and example 5 (prototype) shows that the method of obtaining citric acid according to the invention, in contrast to the method according to the prototype of the obtained culture solution, which contains citric acid and has amylolytic activity.

The result of example 4, in which the fermentation is carried out without adding to the hydrolyzed starch addition of mineral salts in the form of sulphate of zinc, iron (II), copper, shows that after fermentation and separation of the biomass of the fungus obtained culture solution, which contains citric acid and 84.6% and acid-amylolytic enzymes, the activity of which is less than in examples 1-3, in which the use of additional additives called mineral salts.

In example 5 was carried out in conditions of the prototype with the inactivation of biomass - derived culture solution that contains citric acid 85,0%, but has no amylolytic activity.

Thus, the proposed izobreteny the acid but amylolytic enzymes: amylase and glucoamylase, moreover, the conversion of sugars to citric acid is 84,9 and 88.5%, the amylolytic activity of acid-amylase and glucoamylase respectively, a unit of A. C./cm3: of 0.62 to 0.69 and 22.5-28,6.

Sources of information

1. Tokhadze H. C., Kvachadze L,P., Kvesitadze, I. Influence of nutrient medium composition on the synthesis of acid-amylase different Aspergillus species. Applied biochemistry and Microbiology. - M., 1975. - 4. - S. 515-518.

2. RF patent 2132384, IPC 6 12 P 7/48, With 12 N 1/14, 1999.

3. RF patent 975799, MKI 3 12 N 15/00, 1982.

The method of obtaining citric acid, comprising hydrolysis of a starch suspension containing 26 - 30 wt.% dry substances, enzyme drug bacterial-amylase at elevated temperature and pressure, the addition of mineral salts to the hydrolyzed starch, subsequent fermentation of a nutrient medium mushroom - kislotoobrazoutei Aspergillus niger at a temperature not exceeding 32oAnd the Department of culture solution, characterized in that use bacterial-amylase, taken in an amount of 1.5 - 2.0 units amylolytic capacity per 1 g of dry substance of the starch, is added to the hydrolyzed starch addition of mineral salts in the form of sulphates C is the strain VKPM F-171 and after fermentation is separated acidic culture solution, additionally having amylolytic and glucoamylase activity.

 

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