CS200029B1 - Method of activity increase of production strain aspergillus niger at the technical fermentation of citric acid - Google Patents

Description

The present invention relates to increasing the activity of an Aspergillus niger production strain in the technical fermentation of citric acid by growing the strain on an altered nutrient medium.

The Aspergillus niger strain grown on a cultivation pod with the composition used so far gives unstable citric acid extracts, very dependent on the quality of the sweetener. Cooperation is often spore-coined, dark gray conidia, small, conidia carriers low. A considerable amount of malic acid is detected by the chromatographic method both at the citrus and at the end of fermentation. The increase in acidity in citrus fermentation is slow.

The above drawbacks are eliminated by increasing the activity of the Aspergillus niger strain by applying a system of influencing the feeder strain by changing its culture conditions so as to improve the enzymatic activity of the Aspergillus niger fungus, mainly the oxidoreductase activity, and to improve nutrient transport into cells.

This new variety - a nutritional mutant - produces the correct yield of citric acid with the right fermentation technology.

Citric acid is produced by Aspergillus niger. The presence of the secondary acid citrolúhoch suggests that T torr is citric acid as a result of reactions of di- and tri-Carboxylic acid, but at the same time these reakciemi remodeling occurs and the side reactions, for example by condensation of the compounds _Cg and C ^, resulting from cleavage of the glucose molecules. Retko ⁱⁿ these reactions are catalyzed by enzymes relevant. Ifaosity of citric acid and its speed

Thus, the build-up 200,025 is directly proportional to the activity of the enzymes that allow the acid to form by its cycle or by the side-cycle from the side acids.

The major enzymes NAD ⁺ and PAL ·,, which are found in the mitochondria of cells, are lived below for energy. Electron transfer, the donor of which is NADH or succinic acid, forms several complexes. Three of them contain non-heme iron, the fourth firmly bound honey. Movable components allow electron transfer.

DPNH -F _d - Fe —— * Q succinate -> F _g —- * Fe —— »Q

UQH ₂ -e> b - * solv * 0 _χ -► C o - »a —- * Cu * Og where F _d » ^f s - dehydrogenases NADH and succinic acids UQ - ubiquinone - coenzyme Q a, b, c, Cj - cytochromes

The last step is the transfer of electrons to oxygen and the result is the oxidation of the substrate. The inefficient reactions in the Krebs cycle ensure NADH + H ⁺ —e> NAD ⁺ and its careful participation in oxidation. Carbon transfer from reduced nicotinamide dehydrogenases catalyzes flavoproteins. The action of the electrons is iron heme, which changes valence during oxidation.

Simplified tricarboxylic cycle scheme:

Cultvy * Α1 >>

dceivT-KUP + H * /. .

* T. ¹á «» loetovu

A.tu-ufconiiovA in A <->

60th

Madhu

• FAPH «• fltD * to A, ATP <—> 1

ΚοΑ, ΝΑ »*

k.exelj ^ Urove k * »?» *** '' *

^^ (• ΚβΑ

Oxidases β peroxidases have metal / iron, copper / in the prosthetic group. They can be activated by these trace elements. However, in addition to the active enzymatic system, increased transport of substances to the cells is required for good fermentation and rapid metabolite accumulation. The transport is provided by the cytoplasmic membrane. The bed is passive - does not require energy, or active - conditioned by the use of ATP energy generated in the cell. This is an exchange reaction. Upon accumulation of K ⁺ , Mg ⁴⁺ , HgPO 4, H ⁺ and Na ⁺ are released. Sodium ions and potassium ions enter the cell during active transfer, but also during diffusion.

The accumulation of citric acid depends on suitable nutrient crops on which Aspergillus niger is grown. Opaque filings must ensure rapid growth of mold and good cooperation. The composition of the enzyme depends on its composition. The activity of this enzyme system also depends on the culture conditions (temperature, pH, etc.).

Since the energetically important oxidoreductases PAD a, NAD, which affect the production of citric acid by both its cycle and side pathways, have iron and copper in the prosthetic group, these oxidoreductases are activated by iron salts and media. Activation of the enzymes leads to a faster increase in acidity, requiring faster transport of substances (mainly glucose) into cells, which is conditioned by an exchange reaction between sodium and potassium ions through the cytoplasmic membrane. This is achieved by reducing the potassium salt content and adding sodium salts in NaCl or NagSO4. In addition, NaCl is also thermostable and activates some of the enzymes by providing a relatively small diameter positive metal complex, creating a new solution configuration that may be more advantageous over the structural arrangement of some enzyme-substrate complexes. ^{At the} same time, the reduction of phosphorus and magnesium salts has a graceful effect on improving cell transport.

Cultivation is carried out for 9 days at 32 ° C and 1 day at 24 ° C. Conidia are black, large, ⁱⁿ koniddionosiČe high. The conidia obtained under laboratory conditions give stable results in citric acid yield always above 80%. Good results in citric acid yields were also achieved in chambers where the average acid yield. the lemon was 72%. The conidia grown on the new co-opaque soil is characterized by a sharp increase in acidity accompanied by an increase in heat release, which must be removed by the third day of citrus fermentation.

On such technologically correct chambers, on day 9 of citrus fermentation, the citrouss contain citric acid, from minor acids in small amounts gluconic acid and sometimes in minimum quantities malic acid. The average amount of foreign acids determined through calcium citrate is 0.1%. Oxalic acid is not found in lemons.

In order to reduce the amount of gluconic acid, it is recommended to pass through formic acid or glyoerin.

The following table shows the results of the effect of changing the composition of the sporulation pod on the Aspergillus niger activity during fermentation on molasses substrate in hrlenmayer flasks.

Sporulačná pfida % citric acid yield

First / K / original pSda 72.6 Second / K / + Na ⁺ 67.3 Third / K / + Fe ²⁺ 76.3 4th / K / + Fe ²⁺ + Na ⁺ 79.9 5th / K / + Fe ²⁺ + Na ⁺ + Cu ²⁺ - 91.4 - Mg ²⁺ - CN ₂ PO ₄

The results characterize the produced activity of the Aspergillus niger strain grown on various podas. The increase in recoveries of spores obtained on a modified composition pod is on average 17% at 20 replicates, a statistically significant difference.

The operational tests are as follows:

Substrate inoculated with spores 0 acid extract titr. 0 Extract acid cit Aspergillus niger for sugar acid pAutomatic / P / 58.6 56.3 aovými / N / 71.8 71.7

evaluation of conversion results:

- 0- recovery of spores N is higher by 13%

- 0 side acid formation monitored via calcium citrate is 0.1%

- citric acid, low gluconic acid, malic acid only sometimes found in a minimal amount

- mycelium morphology healthy, solid

- from the 4th day of citrus fermentation a sharp increase in acidity, increased heat release.

Claims (1)

OBJECT OF THE INVENTION A method of increasing the activity of an Aspergillus niger production strain in the technical fermentation of citric acid, characterized in that the production strain spores are obtained by cultivation on a soil containing besides the commonly used substances crystalline ammonium sulfate crystalline 2 mg / l, copper sulfate crystalline 0.6 mg / l , sodium chloride 10.0 g / l, while reducing the potassium dihydrophosphate content to 0.06 g / l and the crystalline magnesium sulfate to 0.05 g / l.