EA003211B1 - Method of obtaining of citric acid and its salts thereof by means of periodical continuous fermentation of fungus aspergillus niger - Google Patents

Abstract

1. Method of manufacture of citric acid and its salts thereof by means of fermentation of fungus Aspergillus niger (VKPM) F-718, comprising a stage of preparing of seed grain, a following cultivation of said fungus in nutrient medium, that contains the sources of carbon, nitrogen, phosphorus and microelements, and recovering of target product, characterized that composition, which is used as stuff conidium at the stage of preparing of seed grain, comprises milled brans, peptone milled sugar, milled citric acid, and surfactant species, in particular, Tvin-40 is additionally introduced in cultivation medium, nitrogen and iron quantity is corrected in composition of nutrient medium depending on form of carbon source, pH medium at the initial fermentation stage is established on 4.-5.5. 2. Method according to claim 1, characterized that medium flow velocity at continuous fermentation mode is 0.5-0.6 h<-1>.

Description

The invention relates to the microbiological industry and relates to a method for producing citric acid and its salts, which can be used in the food industry, medicine, pharmacology.

Known methods for producing citric acid and its salts from fermentation solutions obtained by the cultivation of various microorganisms: yeast (RF patent 695230, RF patent 2007459, RF patent 842106, RF patent 2096461), mushrooms (ed. St. USSR 1592336, RF patent 1296580) , bacteria (ed. St. USSR 1221243), a consortium of yeast and fungi (RF patent 2098485). Despite the variety of producers of citric acid in the industrial production of citric acid, the preference is given to mushrooms Akregdshsh shdeg.

Known methods for producing citric acid by cultivation of the fungus Lkregd Shik shdeg on various carbohydrate-containing environments: starch, whey, ethyl alcohol, sugar, molasses (RF patent 213238, Auth. St. USSR 1211290, RF patent 2095416, RF patent 2076906) with periodic, weaning or continuous cultivation (RF patent 2095416, RF patent 907072, RF patent 2076906).

The closest to the claimed invention relates to a method of obtaining citric acid, described in the patent of the Russian Federation 2125607. According to this patent, citric acid is obtained by fermentation of the fungus Akregd Shshdeg VKPM P - 718 on molasses nutrient medium in two stages. In the first stage, molasses is used with a pH of 4.0-5.0, the growth temperature is 32-34 ° C, and after 30-48 hours from the start of cultivation, a continuous supply of molasses and nutrient medium is started at a dilution rate of 0.025-0.03 h ^-1 . In the second stage, a suspension of microorganisms flowing from the first stage is used, the process is carried out at 30-32 ° C and pH 2.0-3.0. The selection of the suspension is carried out at the same speed. Uterine solutions formed during the crystallization of citric acid, is used to further obtain salts of citric acid. When implementing this invention, the concentration of citric acid in the environment is 73-75 g / L. The output from PB 91-95%.

It is known that the synthesis of citric acid is influenced by the cultivation conditions, the quality of the seed, and the composition of the nutrient medium.

The described patent states that growing mycelium on a nutrient medium containing molasses, sucrose, ethyl alcohol and trace elements (Ζη, Co, Mo) contributes to improving the quality of the seed. However, the patent does not provide data on the yield of citric acid before and after growing mycelium under the above conditions. In addition, when growing AkregdShik shdeg mushroom on nutrient media with different carbon sources, the same composition of mineral salts is used, which does not allow creating optimal conditions for mushroom cultivation and their accumulation of citric acid.

The objective of the invention is to intensify the production of citric acid and increase its effectiveness.

The solution of the task is provided through the use of technological methods that improve the quality of the seed and optimize the cultivation conditions and the release of citric acid, which allows, under periodic fermentation, to increase the yield of citric acid and, in continuous mode, increase the flow rate to 0.05 - 0.06 h ^-1 .

The essence of the invention is as follows. At the stage of preparation of the seed material with the aim of improving its quality, a conidia filler containing crushed bran, peptone, powdered sugar, crushed citric acid is used. The use of this conidium filler not only ensures the safety of spores, but also contributes to the increase of their biochemical activity. The use of this filler, along with a reduction in the consumption of conidia, contributes to an increase in the biosynthesis of citric acid by 7% and an increase in the activity of germination (germination) of conidia by 15%.

At the stage of fermentation, in order to create optimal mineral composition of cultivation conditions for the fungus Akregd Shik Shdeg and increase the yield of citric acid, nitrogen is introduced into the culture medium in the form of ammonium salts, ferrous sulfate and non-ionic surfactant (Tween-40). The optimal amount of salts of nitrogen and iron on the medium with sugar moths is 1 g / l and 8-9 g / l, respectively, and on the medium with molasses 2 g / l and 10 -15 g / l, respectively. The amount of surfactant is 120-170 g / l. Fermentation is carried out at an initial pH of 4.5-5.5. Optimization of the mineral composition, pH of the medium and the introduction of surfactant into the culture medium increases the yield of citric acid by an average of 12%.

The use of the above-described technological methods in the continuous mode of cultivation allows you to increase the flow rate of the medium to 0.05-0.06 h ^-1 . The citric acid obtained during the fermentation stage (crystalline or purified production solutions) is then sent to obtain citric acid salts (sodium citrate or potassium citrate).

A solution of alkali is loaded into a reactor equipped with a stirrer, a cooling coil, a PH meter, a thermometer (40% solution α or 35-40% KOH solution). Then, after turning on the mixer and supplying water for cooling, 50-60% solution of lemon Ki3 slots is fed to the reactor. Neutralization is reduced to pH = 7.27.6 (in the case of ΝαΟΗ) or pH 7.8-8.0 (using KOH), while not allowing an increase of 1 ° to 80 ° C.

The process of evaporation and crystallization is carried out in a vacuum crystallizer, which is a vertical apparatus equipped with a stirrer, thermometer, vacuum gauge. The residue is carried out at 0.8-0.9 at, not allowing the temperature to rise above 80 ° C.

When reaching the density of the evaporated sodium citrate solution to 1.48-1.54 kg / dm ³ (potassium citrate to 1.58 kg / dm ³ ), the process is completed and transferred to crystallization.

At the same time, water is supplied to the coil and the mass is cooled to 1 ° = 42-45 ° (or to 20 ° C). You get three-substituted 2-aqueous sodium citrate (or three-substituted 1-aqueous potassium citrate).

The obtained crystals are separated from the mother liquor in a filtration centrifuge with continuous discharge of sediment and dried in a fluidized bed dryer. This is followed by sieving of crystals into fractions; non-standard crystals are returned to recrystallization. The product is packaged in paper bags.

Example 1

The seed was prepared as follows: dry conidia were mixed in a 1: 1 ratio with the filler of the following composition:

crushed bran - 50%;

peptone - 35%;

crushed sugar -14%;

crushed citric acid - 1%.

To obtain a homogeneous inoculum, conidia with a filler was mixed for 60 hours. The germination rate of this inoculum was 95% (in the control variant, 80%).

Mushroom Lcr.shdeg grown on medium with molasses:

Molasses - 2% (by PB);

(ΝΗ4) 2§Ο4 - 2 g / l;

KN ₂ PO ₄ - 0.2 g / l;

Μ§8Ο4 · 5Η2Ο - 0.25 g / l;

2po ₄ 8O ₄ -7H ₂ O - 0.005 g / l;

Ρе8Ο ₄ · 7Η ₂ Ο - 15 g / l.

pH was adjusted to 4.5. Additionally, surfactant (tween-40) in the amount of 170 mg / l is added to the culture medium.

The cultivation is carried out under continuous conditions in 2 serially connected fermenters.

I stage is carried out at the initial value of pH = 4.5 and 1 ° = 32 ° C. After 26 hours from the start of fermentation, after reducing the intensity of mycelium formation, the process is transferred to a continuous mode by continuously feeding molasses solution (containing PB8%) and nutrient salts at a rate of D = 0.05 h ^-1 to the medium. Flowing suspension with the same speed enters the second stage (the second fermenter), where the fermentation is carried out at a temperature of 30 ° C. The pH value in the second stage is reduced to 2.0. The concentration of citric acid in the medium is 86 g / l.

Example 2

Same as in example 1, but the cultivation of lead on the medium of the following composition:

Sugar - 95 g / l;

(ΝΠ4) 28Ο4 - 1 g / l; KH2PO4 - 0.2 g / l;

\ 1d8O% 11 <) - 0.25 g / l;

ΖηδΟ ^^ - 0.005 g / l;

Ρе8Ο ₄ · 7Н ₂ Ο - 8.5 g / l;

pH of 5.5.

The concentration of citric acid is 79 g / l.

Example 3

Same as in example 1, but tween-40 in an amount of 120 mg / l is added to the culture medium. The flow rate is D = 0.06 h ^-1 .

The concentration of citric acid is 90 g / l.

Example 4

Same as in example 1, but the process is conducted in periodic mode for 7 days.

The concentration of citric acid in the medium is 110 g / l.

Example 5

A 40% ΝαΟΗ solution and 60% citric acid solution are fed to the jacketed reactor until the pH of the medium is 7.5. The resulting solution was evaporated at 0.9 atm to a concentration of sodium citrate of 1.5 kg / l. The resulting solution is cooled to a temperature of 45 ° C. The precipitated crystals of three-substituted sodium citrate are separated in a centrifuge and dried.

Example 6

A 35% KOH solution and a 50% citric acid solution are fed into the reactor until the pH of the solution is set to 8.0. The resulting solution was evaporated at 0.8 atm to a concentration of potassium citrate of 1.58 kg / l. Next, the solution is cooled to a temperature of 20 ° C. The precipitated crystals of potassium citrate are separated by centrifugation and dried.

Claims (2)

1. The method of production of citric acid and its salts by fermentation of the fungus AkregdShik shdek VKPM R-718, which includes the stage of preparation of seed, the subsequent cultivation of the specified fungus in a nutrient medium containing a source of carbon, nitrogen, phosphorus and trace elements, and the selection of the target product, characterized in that at the stage of preparation of the inoculum as a filler of conidia, a composition is used, including crushed bran, peptone, crushed sugar, crushed citric acid, in a culture medium I additionally introduce a non-inogenic surfactant, in particular, Tween-40, the amount of nitrogen and iron salts is adjusted in the composition of the nutrient medium depending on the type of carbon source, the pH of the medium at the initial stage of fermentation is set to 4.5-5.5. 2. The method according to claim 1, characterized in that the flow rate of the medium during continuous fermentation is 0.05-0.06 h ^-1 .