The method of obtaining citric acid

 

(57) Abstract:

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 method consists in the fact that at a temperature of 32°C from the fermented solution is separated mycelium of the fungus-cyclotourists Aspergillus niger, purify the obtained culture fluid by sequential filtration or adsorption of impurities, and then divide by ultrafiltration through a membrane, retaining the molecular weight within 1000-50000, on the solutions of target products. The method allows to simultaneously obtain two target product: purified citric acid solution and the complex kislotostabilen amylolytic enzymes. table 1.

The invention relates to the microbiological industry and relates to method of producing citric acid by fermentation medium on the basis of the hydrolyzate of starch by the fungus Aspergillus niger and release it from the fermented solution.

The traditional method of obtaining crystalline citric acid includes many appropriate solution, the precipitation of the desired product in the form of calcium citrate, the decomposition of the salt with sulfuric acid, filtering the gypsum slurry through active carbon, crystallization [1].

The use of environmentally harmful chemicals significantly complicates the process and requires additional cleaning solutions of citric acid, regeneration of the sorbent, the use of additional reagents, increasing energy and material costs. In addition, the method does not provide the possibility of allocating kislotostabilen enzymes.

A method of obtaining citric acid in liquid form with the use of bentonite and zeolite for treatment of acid solution from the remnants of the mycelium and the subsequent concentration [2]. The acid concentrate is characterized by a specific color and the presence of up to 0.5% of the protein substances. Subsequent separation of the impurities in no way.

The known method for preparation of homogeneous-amylase from the culture liquid after the fermentation of a nutrient medium thermophilic strain Century stearothermophilus. The filtrate of the culture fluid, containing 5.0% AU/mg protein, concentrated by ultrafiltration before selecting the solution with the activity of 10.6% AU/mg protein, then spend the OS is related enzymes admixtures. The yield of pure product-amylase is 22,1% [3].

In the allocation method of the enzyme ultrafiltration method allows the concentration of culture filtrate of liquid without phase transformation at room temperature and while exemption from ballast substances (pigments, nitrogen and other low molecular weight compounds).

However, the known method does not provide for the obtaining and isolation from the culture fluid of the two target products: low molecular weight compounds and enzyme complex.

A method of obtaining citric acid, including the fermentation of carbohydrate-containing raw material, the separation of the mycelium of the fungus Aspergillus niger, cleaning fermented solution suspensions of activated bentonite, followed by the separation of solution and processed with activated carbon fiber material brand OUT-M, Busofit-T Busofit-TM or Carbon-asset, having a volume of sorption time limit 0,30-0,80 cm3/g, taken in an amount of 2.0 to 12.0 g per 1 DM3solution.

The result is purified citric acid solution not containing proteins, including enzymes, acidity of 14.6-14.7 per cent, which is 3.3 to 4.0% below pointof as the target product in no way, since the separation of the mycelium is carried out at temperatures above 70 C, is the inactivation of enzymes and coagulation of proteins that become unwanted impurity.

The closest present invention is a method for acid phosphatase from the culture fluid containing citric acid and having the activity of phosphatase by ultrafiltration through a membrane, retaining the molecular weight phosphatase from 5000 to 50000, at a temperature 5-50 ° C, a pressure of 0.1-0.6 MPa, flow rate through the membrane 4-60 kg/m2·hours After separation by ultrafiltration get the solution with the activity of phosphatase 5-50 Kat·1-1and concentrate having the activity of a phosphatase 1500-50000 Kt·1-1[5].

Thus, the known method [5] is obtained from the culture liquid of the citric acid solution, is not purified from active enzymes, and concentrate of acid phosphatase. The citric acid solution having an activity of 5-50 Kat·1-1phosphatase, requires further allocation of impurities to obtain pure acid solution.

The technical result of the invention is the selection of fermentive solution after fermentation medium on the basis of gidrol mikoliticheskikh enzymes.

The technical result of the invention is achieved by a method of obtaining citric acid from the culture fluid, including the separation of the mycelium of the fungus-cyclotourists from fermented solution, the separation of the fermented solution by ultrafiltration through a membrane, retaining the molecular weight of the enzyme in the citric acid solution and the solution of the enzyme, which according to the invention is used with the activity of-amylase and glucoamylase culture liquid, separating the mycelium of the fungus-cyclotourists Aspergillus niger at a temperature of 32C, to the division of clean fermentary solution with a temperature of 32C using filtering or absorbing means, moreover, using a membrane with a pore size of 0.65 μm or suspension of bentonite or carbon fiber material of the type Carbon-asset, then share purified fermented solution through a membrane, retaining the molecular weight of 1000-50000, on the solution of the target product and solution kislotostabilen amylolytic enzymes.

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

In the examples used fermented R is the culture fluid [6].

Fermented solution is a liquid, the concentration of citric acid which is 100-150 g/DM3that is getstringarray activity (Dak) - (0,5-2,5) units/cm3, saharaganj activity (Nao) - (a-20.0 to 90.0) units/cm3pH from 1.3 to 2.8, the optical density Dmax400 nm to 1.9 to 2.5,max750 nm of 2.0 to 3.0, which characterize, respectively, the color and turbidity of the solution, the protein content of 1.5 - 5.0 g/DM3.

The examples use the fermented solution having a concentration of citric acid 125 g/DM3The duck is 1.8 units/cm3Sak 58 units/cm3Dmax400 nm 2,4, Dmax750 nm 2,34 containing 3.5 g/DM3protein.

As filtration media using a membrane with a pore size of 0.65 μm, as absorptive means is activated bentonite suspension containing bentonite 10 wt.%, or carbon fiber material Carbon asset.

Filter and absorptive means are produced by domestic and foreign industry. The examples use a filtration unit “Sartokan” company Sartorius AG, Germany, is equipped with a standard and minimalrome Sartopure PP 2559, in particular 559 15 05 P7 with a retention rate of 0.65 μm. For ultrafiltration applications(UMM): SAR-50 50·103, Mifil-20 PA 20·103hollow fiber - 15 PS 15·103, Mifil - 10 PA from 10103hollow fibers 5 from PS 5103hollow fiber - 1 PS 1103. Feature of the device: the operating pressure of 0.1-0.2 MPa, flow rate - 20-37 DM3/m2·h

In the examples using known methods of analysis: determination of acid solutions titrimetric method determination of the optical density of the solutions, getstringarray and saharaganj activity using the colorimetric method, the protein by the method of Lowry.

Example 1.

In a flask with a volume of 750 cm3placed 50 cm3the nutrient medium and seeded her grown mycelia. The flask is placed on the rocking chair with the speed of 160 min-1and incubated for 6 days at a temperature of 32C. After fermentation the biomass of the fungus is separated in a Buechner funnel and fermented solution determine the optical density of the solution, active kislotostabilen amylolytic enzymes and the concentration of citric acid.

Fermented solution has a volume of 2.5 DM3having a temperature of 20 ° C, passed through a filtration membrane to remove residual mycelium, and then the solution containing the acid and the scrap UMM 50,0103the acid solution and the solution kislotostabilen amylolytic enzymes.

The results of the experiment are shown in the table.

Example 2.

Fermentation and purification of fermented solution from the residue of mycelium carried out according to example 1.

The separation of a solution with a temperature of 15 ° C is performed by ultrafiltration through a membrane Mifil - 20 PA with a limit UMM 20,0103um.

The results of the experiment are shown in the table.

Example 3.

Fermentation and separation of the mycelium carried out analogously to example 1.

Fermented solution of 0.5 DM3with a temperature of 20 ° C is passed through the filter membrane to remove residual mycelium, and then the solution containing citric acid and cyclotosaurus amylolytic enzymes, separated by ultrafiltration through a membrane hollow fiber - 15 PS with a limit UMM 15103.

The data are shown in the table.

Example 4.

Fermentation, separation of the mycelium and cleaning fermented solution through the filter membrane is carried out analogously to example 3.

Fermented solution with a temperature of 32C, containing citric acid and cyclotosaurus amylolytic the x2">

The data are shown in the table.

Example 5.

In the conditions of example 1 to fermented solution has a volume of 2.5 DM3add 2 g/DM3pre-prepared activated suspension containing bentonite 10 wt.% and incubated the mixture for 40 min, after which the precipitate is separated and the solution containing the two target product, is subjected to ultrafiltration through the membrane separating Mifil - 10 PA with a limit UMM 10,0103the acid solution and the solution of complex enzymes.

The data are shown in the table.

Example 6.

Fermentation, separation of the mycelium carried out analogously to example 1.

Cleaning fermented solution of 0.5 DM3with a temperature of 25C carried out by passing it through a carbon sorbent, which is used as the non-woven fabric material brand Carbon-asset, having a volume of sorption long 0,80 cm3/g and taken in an amount of 2.0 g per 1 DM3solution. The purified solution, 0.5 DM3containing acid and a complex of enzymes, separated by ultrafiltration through a membrane hollow fiber - 1 PS with a limit UMM 1,0103the acid solution and the solution of complex kislotostabilen and

Obtaining fermented solution is carried out analogously to example 1. Fermented solution with a volume of 1000 cm3containing 125 g of citric acid and having the activity of a-amylase of 1.8 units/cm3and glucoamylase 58 units/cm3, is subjected to ultrafiltration at a temperature of 20C through the membrane Mifil - 20 PA with retention of molecular weight of 20,000, with an operating pressure of the installation of 0.2 MPa, flow rate 25 DM3·m-2·h-1and receive 940 cm3slightly coloured filtrate containing citric acid and protein impurities of 1.9 g/DM3and 60 cm3concentrated solution kislotostabilen enzymes with activity units/cm3, -amylase 28,3, glucoamylase 570.

The data are shown in the table.

The indicators of the quality of the fermented solution, after treatment containing acid and enzymes, and solutions after separation are presented in the table in examples 1-7.

Comparison of the results obtained in examples 1-6 of the proposed method in example 7 - the prototype shows that the use of examples 1-6 fermented solution with a temperature of 32C allows after cleaning residues from mycelium using filtering or absorbing means get the which then using ultrafiltration through a membrane with retention of molecular weight in the limit values from 1.0 to 50.0)·103is divided into the acid solution and the solution kislotostabilen amylolytic enzymes as target products.

In example 7 (prototype) fermented solution separated by ultrafiltration and get the solution containing citric acid and mixtures of proteins, as well as the solution of amylolytic enzymes. Impurities protein substances - 1,9 g/DM3increase the chroma (D4000,58) and turbidity (D7500,120) solution of citric acid, lowers the quality of it, cause the need for further purification of the acid solution.

Thus, the proposed method from fermented solution after fermentation medium on the basis of hydrolyzed starch mold fungus Aspergillus niger receive two of the target product is pure citric acid solution and the solution of complex kislotostabilen amylolytic enzymes.

SOURCES of INFORMATION

1. Instruction manual for the production of citric acid. -L.: KNEIPP, 1981, 130 S.

2. RF patent №2129612, IPC 6 12 P 7/48, 1999.

3. Grachev, I. M. Technology of enzyme preparations. - M.: Agropromizdat, 1987, S. 177.

4. RF patent №2159286, IPC 6 12 P 7/48, 2000.

5. Patent No. 275986, IPC 5 12 P 7/48, 1992.

C until a clear separation fermented solution with a temperature of <32C using filtering or absorbing means, and use a membrane with a pore size of 0.65 μm or suspension of bentonite, or carbon fiber material Carbon-asset, and then share purified fermented solution through a membrane, retaining the molecular weight within 1000-50000, on the solution of the target product and solution kislotostabilen amylolytic enzymes.

 

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FIELD: cleaning oil from contaminants by crossing micro-filtration.

SUBSTANCE: proposed method includes circulation of oil above surface of reinforced membrane which is supported by tube provided with open pores and made from fiberglass plastic, carbon fiber reinforced plastic or organic plastic. Micro-filtration of oil is performed through fluropolymer membrane filled with kieselguhr or bentonite at size of pores from 0.1 to 0.5 mcm. Method is realized at rate of 2-3 m/s under pressure of 0.05-0.3 Mpa at temperature of 20-60°C.

EFFECT: enhanced efficiency of cleaning oil.

3 cl, 1 dwg, 1 tbl, 11 ex

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