Method for preparing calcium citrate

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 allocation of citric acid from the culture solution after fermentation by Aspergillus niger culture media based on hydrolyzed starch, in particular allocation method of citric acid in the form of a salt, and the receipt of enzymes.

There is a method of allocating salt of citric acid, calcium citrate is that after fermentation by Aspergillus niger culture liquid is filtered from biomass on a suction filter. Lighten the filtrate with activated charcoal. The charcoal is filtered off and washed with demineralized water. The washing water and the filtrate is neutralized 2 h at 80°C, filtered on a suction filter. Washed with hot demineralized water, the precipitate of calcium citrate on the filtrate to a negative reaction of wash water to the sulfates and chlorides. The precipitate is dried at 60°C. Receive 16820 g of calcium citrate tetrahydrate jet clean /1/. The release of salt from theoretical reaction is of 62.3%. After separation of the calcium citrate get citratesildenafil the filtrate, which is a waste product and is exposed to various types of recycling. Method /1/ not available get from nitratsoderzhaschie filtrate kislotostabilen amylolytic enzymes.

There is a method of allocating the product is homogeneous α -amylase from the culture liquid after the fermentation of a nutrient medium thermophilic strain .stearothermophilus. The filtrate of the culture fluid, containing 5.0 IU AU/mg protein, concentrated by ultrafiltration before selecting the solution with the activity of 10.6 IU AU/mg protein, followed by precipitation of the enzyme complex with ethanol, drying the precipitate, the subsequent deep cleaning of the drug from related enzymes impurities. The yield of pure drug α-amylase is 22,1% /2/.

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 compounds).

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

The closest present invention is a method of obtaining calcium citrate from solutions after fermentation, containing 117 g/DM3citric acid, comprising mixing citric acid solution and the solution of one of the compounds of calcium: calcium hydroxide, calcium oxide, calcium carbonate, containing in the calculation of the calcium oxide 182 g/DM3in techenie more than 1 sec in the mixer, then the exchange reaction with the formation of a supersaturated solution of calcium citrate at a temperature of 20-50°C for 20 minutes and pH 4.5-5.9 in the wind tunnel, the precipitation of calcium citrate at a temperature of 60-90°and a pH of 5.4 to 6.5 for up to 2 hours in one or a series of two boilers /3/.

As a result of continuous process get calcium citrate, the purity of which was confirmed by tests:

a) the optical density of the solution obtained after decomposition of calcium citrate with sulfuric acid;

b) the behavior is relatively concentrated sulfuric acid /3/.

The possibility of obtaining and allocating kislotostabilen amylolytic enzymes in no way.

The technical result of the invention is the selection from the culture solution after fermentation by Aspergillus niger environment on the basis of the hydrolyzed starch is an additional product: complex kislotostabilen amylolytic enzymes.

The technical result of the invention is achieved by a method of producing calcium citrate containing citric acid culture solution after fermentation of the fungus Aspergillus niger, which includes adding calcium to the culture solution, the precipitation of calcium citrate in an acidic environment and the Department of calcium citrate from the solution, which according to the invention of IP is result with α -amylase and glucoamylase activities of the culture solution to which is added a suspension of calcium containing in the calculation of the calcium oxide 0,22-0,28 kg/DM3to a pH in the range of 3.2-5.9 and at a temperature in the range of 10-50°, citrate precipitated under these conditions within 30-60 minutes, then after separation of the target product get the solution of complex kislotostabilen amylolytic enzymes.

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

The examples use the culture solution, which is obtained by fermentation medium on the basis of the hydrolyzate of starch by the fungus Aspergillus niger and filtering the culture fluid /4/.

The culture solution is a liquid, the concentration of citric acid which is 100-150 g/DM3that is α-amylase activity (AU) - (0,5-2,5) u/cm3, glucoamylase activity (FPP) (20,0-90,0) u/cm3pH from 1.3 to 2.8. In the examples using a culture solution having a concentration of citric acid 127 g/DM3AC 2.5 u/cm3, Voice 90 IU/cm3pH of 2.0.

To highlight calcium citrate use milk of lime, which is produced in the form of a suspension with a density of 1.16-1.20 g/DM3and containing in the calculation of the calcium oxide 0,22-0,28 kg/DM3WM is ansii.

In addition to the milk of lime to precipitate calcium citrate can be used compounds such as chalk, calcium oxide.

In the examples using known analysis methods: colorimetric method for determination of AU and Voice solutions, titrimetric method for determination of acidity of solutions, tests for purity of calcium citrate, chelatometric method for determining the mass fraction of calcium citrate.

Example 1.

To culture solution after fermentation by Aspergillus niger volume 1 DM3containing 127 g of citric acid and having a pH of 2.0, α-amylase activity of 2.5 u/cm3and glucoamylase activity 90 IU/cm3, add the lime milk suspension density ρ 1,16 g/cm3at a temperature of +10°With up to a pH of 3.2, maintain the reaction mixture at a temperature of +10°and pH of 3.2 within 60 minutes. The precipitate of calcium citrate is separated in a Buechner funnel.

In the separated solution determine the acidity (%) titrimetric method, the enzymatic activity of the AU and Voice colorimetric method.

The precipitate of calcium citrate washed with water with a temperature of 50-60°until the pH of wash water of 5.8 to 6.0. Dried at a temperature of 105°receive calcium citrate, mass fraction which is 99,29% (based on absolute dry substance.

The results are presented in t the blitz.

Example 2.

To the culture solution of 1 DM3content of citric acid 127 g AC 2.5 u/cm3, Voice 90 IU/cm3, pH 2.0 and add the lime milk suspension with a density of ρ 1,16 g/cm3at a temperature of +50°With up to a pH of 3.2, maintain the mixture at a pH of 3.2 and a temperature of +50°C for 30 minutes, then the precipitate is separated on a Buechner funnel.

In the separated solution determine the acidity and activity kislotostabilen amylolytic enzymes.

The precipitate of calcium citrate washed with water at a temperature of 50-60°C, dried at 105°S, as in example 1.

The results are presented in the table.

Example 3.

To the culture solution of 1 DM3content of citric acid 127 g AC 2.5 u/cm3, Voice 90 IU/cm3, pH 2.0 was added a suspension of chalk with a density of ρ 1,17 g/cm3at a temperature of +20°With up to a pH of 4.5, maintain the reaction mixture at pH 4.5 and a temperature of +20°C for 45 minutes, then the precipitate is separated on a Buechner funnel.

In the separated solution determine the acidity and activity kislotostabilen amylolytic enzymes.

The precipitate of calcium citrate washed with water at a temperature of 50°C, dried at 105°S, as in example 1.

The results are presented in the table.

Example 4.

To the culture solution of 1 DM3with the content of what W citric acid 127 g, AC 2.5 u/cm3, Voice 90 IU/cm3, pH 2.0 and add the lime milk suspension with a density of ρ 1.19 g/cm3at a temperature of +10°With up to a pH of 5.9, then maintain the reaction mixture under these conditions for 30 minutes, after which the precipitate is separated by filtration.

In the separated solution of a complex of enzymes determine the acidity and activity of the speakers Voice.

The precipitate of calcium citrate washed with water at a temperature of 50-60°C, dried at 105°S, as in example 1.

The results of the experiment are shown in the table.

Example 5.

To the culture solution of 1 DM3content of citric acid 127 g AC 2.5 u/cm3, Voice 90 IU/cm3, pH 2.0 and add the lime milk suspension with a density of ρ 1.19 g/cm3at a temperature of +50°With up to a pH of 5.9, maintained at these conditions the reaction mixture for 60 minutes, after which the precipitate is separated by filtration.

In the separated solution of a complex of enzymes determine the acidity and activity of the speakers Voice.

The precipitate of calcium citrate washed with water at a temperature of 50-60°C, dried at 105°S, as in example 1.

The results of the experiment are shown in the table.

Example 6 (the prototype).

To the culture solution, as in example 1, add the milk of lime containing in the calculation of the calcium oxide of 0.182 kg/DM3(ρ 1,184 g/cm3), PR is the temperature of +50° With up to a pH of 5.9, and then incubated the mixture at a temperature of 70°and a pH of 5.9 for 120 minutes, after which the precipitate of calcium citrate is separated by filtration.

In the separated solution determine the acidity and enzymatic activity.

The precipitate of calcium citrate dried at 105°S, as in example 1.

The results of the experiment are shown in the table.

Table
№ p/pConditions of deposition of calcium citrate from the culture solution at AC 2.5 u/cm3, Voice 90 IU/cm3The release of calcium citrate from theoretical %The characteristic solution of the enzyme complex
Acidity, %Enzymatic activity
pHTemperature, °Time min
AUGLS
u/cm3%u/cm3%
13,2106044,31,302,1485,675,8784,3
23,250 3050,10,491,8975,666,8074,0
34,5204547,60,492,3995,6to 84.7094,1
45,9103051,00,172,4297,087,3097,0
55,9506057,20,072,2891,280,7089,6
6(the prototype)
5,97012061,20,05NoNo

The comparison presented in table results of examples 1-5 according to the invention, and example 6 of the prototype shows that from the culture solution after fermentation by Aspergillus niger containing citric acid and having the enzymatic activity of the AU and Voice, the method according to the present invention receives citrate cal the Oia with access from theoretical 44,0-57.2 per cent and complex kislotostabilen amylolytic enzymes: α -amylase and glucoamylase with access 74-97%.

In the conditions of example 6 (prototype) received calcium citrate (output 61,2%), but from the culture solution as in examples 1-5, in the conditions of example 6 is not received by the enzymes, since at temperatures above 70°and exposure times of 120 minutes, the solution loses its enzymatic activity.

Thus, the method according to the invention, obtained two products: calcium citrate and solution of complex kislotostabilen amylolytic enzymes. Both products can be used in the food industry.

Sources of information

1. RF patent №2132878, IPC712 P 7/40, 12 P 7/48, 1999.

2. Grachev IM Technology of enzyme preparations. M: Agropromizdat, 1987. S.

3. The application of Germany 3014503, MKI307 With 59/265, 1981.

4. RF patent №2132384, IPC612 P 7/48, C 12 N 1/44, 1999.

The way to obtain calcium citrate containing citric acid culture solution after fermentation of the fungus Aspergillus niger, which includes adding calcium to the culture solution, the precipitation of calcium citrate in an acidic environment and the Department of calcium citrate from solution, characterized in that is used with α-amylase activity in the range of 0.5 to 2.5 units/cm3and glucoamylase activity within 20-90%/cm3the culture solution to which is added a suspension connection is in calcium, contains per calcium oxide 0,22-0,28 kg/DM3to pH in the range of 3.2-5.9 and at a temperature in the range of 10-50°, citrate precipitated under these conditions for 30-60 min, then after separation of the target product get the solution of complex kislotostabilen amylolytic enzymes.



 

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