RU2078810C1 - Strain of fungus aspergillus niger - a producer of citric acid - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: new strain A. niger ВКПMF-696 - a producer of citric acid is obtained after selection from the strain storing in Museum ВНИИ ПАКК. Strain ВКПMF-696 forms round, cream-color conidia, mean size of conidium is 3.8 mcm, sterigmas are bilayer, their length - 12.5 and 6.8 mcm. Strain ferments sucrose-mineral nutrient media and media obtained after starch hydrolysis. Strain shows the stable resistance to acid-formation and high yield of citric acid (82.8-90%) from sugar during 1 year strain storage. EFFECT: increased yield of the end product. 2 tbl

Description

 The invention relates to the microbiological industry and the selection of Aspergillus niger fungus producer of citric acid for deep fermentation of sucrose-mineral media and prepared from starch hydrolysates.

Known mutant IBR 1 for the cultivation of Aspergillus niger on sucrose-mineral media, fermenting the nutrient medium for 14 days with a sugar yield of 73-82% [1]
The disadvantage of this strain is the relatively low yield of citric acid with a long duration of fermentation.

 The invention is directed to a strain forming light-colored conidia with stable acid formation activity during long-term storage of the strain and a high yield of citric acid from sugar during fermentation of sucrose-mineral media and media prepared from starch hydrolysates.

 The new strain is selected from black-colored strains of Aspergillus niger, preserved in the collection of the All-Russian Research Institute of PACK. At various doses of ultraviolet radiation, 55 color mutants were selected from the culture of the museum strain, having brown, beige, cream color conidia.

 Of these, the strain with the creamy color of conidia and the light beige surface of the colony was distinguished by the most stable and high activity.

When cultivated on a sucrose-mineral medium in deep conditions, the new strain forms citric acid with a yield of spent sugar of 87.6-90%, which significantly exceeds the prototype strain VKP MF-501 (Table 1). When fermenting starch hydrolysates, the sugar yield of the new strain is quite high and is 82.8%
A significant advantage of the new strain is stably stable acid formation during its long-term storage. Compared with the prototype, the new light-colored strain retains constantly high producing activity - after 12 months of conidia storage, the yield of citric acid upon fermentation of the sucrose-mineral medium is 88.7% per 1 dm ³ / day, 23.7 g of citric acid is formed. At the same time, the prototype VKPMF-501 for 12 months of storage reduces the yield of citric acid from sugar from 82% to 65.25%; the formation of citric acid by 1 dm ³ / day decreases from 21.9 g to 17.4 g.

 The new selection strain Aspergillus niger in the All-Russian collection of industrial microorganisms has been assigned a collection number VKPMF-696.

 The cultural and morphological properties of the new strain Aspergillus niger VKPMF-696 are as follows.

On the 5th day of growth on wort agar at 32 ^o C, a giant colony with a diameter of 90 mm. The colony is light beige. The center of the colony is elevated. In a radius of 10 mm, the conidiomy is abundant; on the rest of the colony, conidial heads are formed in groups, not very densely. A “sterile” margin with immature conidial heads is about 10 mm in radius. After 7 days, conidia mature on the entire surface of the colony. The mycelium is dense from the sole in the center of the colony, the rest of the mycelium is thin.

 After 10 days of cultivation, conidial heads with a diameter of 64-225 microns, an average of 119 microns. Sterigma of the first layer is 12.5 μm (as they age, they increase in size, becoming a bubble); sterigma of the second layer - 6.8 microns.

 Conidia are rounded with a cream shell, the average diameter of the conidia is 3.8 μm. Conidiophores are straight, their length ranges from 270 microns to 1800 microns, an average of 870 microns. The width of the section of the conidiophore from 9 to 18 microns, an average of 13.5 microns. Conidiophores unpainted.

 The new strain differs from the well-known beige strain BKPMF-501 in larger conidia (VKRM-501 strain has a conidia diameter of 3.4 μm) and light beige in color.

Strain VKRM-696 identified by the definition of fungi of the genus Aspergillus [2]
Physiological and biochemical properties
Strain Aspergillus niger BKPMF-696
Aerob, cultivation temperature 28-32 ^o C, pH -5.5.

 Attitude to carbon sources: assimilates glucose, fructose, sucrose, maltose, to a lesser extent mannitol, galactose, sorbitol.

 Attitude to nitrogen sources: assimilates nitrogen of organic compounds, for example protein, peptone, amino acids, yeast autolysate, urea, assimilates ammonium salts, nitrates.

 Genetic feature of the prototroph strain.

Save method. The new strain is stored in the form of air-dried conidia, separated from the fungus culture grown on wort agar for 10 days (6 7 days at 32 ^o C and 3 4 days at room temperature). Storage of conidia at a constant room temperature, relative air humidity not exceeding 75% are protected from direct sunlight.

Example 1. For laboratory testing, seed is grown in test tubes on wort-agar; for semi-production tests, the seed is grown in cuvettes on a solid nutrient medium, prepared on the basis of beer un hopped wort containing 7% dry matter and compacted with agar-agar (20 g / dm ³ ), pH 5.8.

The height of the medium layer is 12 13 mm. The sterile medium is inoculated with conidia of the fungus culture and incubated in a thermostat at 32 ^o C for 7 days. The relative humidity in the thermostat gradually decreases: the first four days 80% 60% the last three days 60% 40% For complete maturation and drying, the test tube culture of the fungus is kept at room temperature for 2 days. After 7 days, conidia are removed from the surface of the mushroom films in cuvettes using a special vacuum device. Wet conidia are dried to a residual moisture content of 5-8%
Store at room temperature.

The laboratory fermentation process is carried out in-depth on a rocking machine АВУ 50p with a number of swings of 160-180 in 1 min. The air temperature in the rocking room is 32 ^o C. The nutrient medium is prepared in flasks with a capacity of 750 cm ³ ; volume of medium 50 cm ³
A fungal culture grown on wort agar for 10 days, or air-dried conidia (10 ⁷ conidia / cm ³ medium) inoculate a nutrient medium and inoculate mycelium for two days. The main raw material for the preparation of media in this example is granulated sugar.

The composition of the medium for producing seed mycelium (g / dm ³ ): sugar 50, ammonium nitrate 2.5, magnesium sulphate 0.25, potassium dihydrogen phosphate 0.16, beet molasses 17 (to stimulate growth), tap water up to 1 dm ³ pH 5.0 5.5. Growing mycelium in an amount of 10 cm ³ sow 50 cm ^{3 of} fermentation medium.

The composition of the fermentation medium differs from the composition of the nutrient medium in the sugar content increased to 150 g / dm ³ . In total, 8.0 g of sugar is spent on cultivating seed mycelium and fermentation. The media are sterilized in an autoclave by fluid steam for 15 minutes and at 0.05 MPa for 30 minutes.

Over 5 days of fermentation, strain BKPMF-696 formed 7.01 g of citric acid, the removal of citric acid was 23.4 g / (dm ³ • day); yield of citric acid from introduced sugar 87.6% biomass productivity 15.2 g of citric acid per 1 g of air-dried biomass. In the known strain BKPMF-501 (control experiment), these indicators are significantly inferior (table. 1).

Example 2. The seed material of a new strain is harvested according to the method described in example 1. The raw material for the preparation of nutrient and fermentation media is a starch hydrolyzate with a glucose content of 74.9%. The fungal seed is grown on an AVU 50p rocking chair for two days on a composition medium, g / dm ³ : hydrolyzed starch 67; ammonium nitrate 2.5; potassium dihydrogen phosphate 0.16; magnesium sulfate seven-water 0.25; zinc sulfate 0.005; tap water up to 1 dm ³ .

Sowing mycelium in an amount of 10 cm ³ contribute to the fermentation medium of the composition, g / DM ³ : starch hydrolyzate 94; ammonium nitrate 1.6; potassium dihydrogen phosphate 0.16; magnesium sulfate seven-water 0.25; zinc sulfate 0.005. The initial volume of the fermentation medium is 50 ml. After two days, the fermentation medium is added in two doses with a concentration of 250 g of sugar per 1 dm ^{3 a} solution of starch hydrolysis in an amount of 24 cm ³ .

The total consumption of sugar per flask is 10 g. The nutrient and fermentation media are autoclaved for 15 minutes with steam and 20 minutes at 0.05 MPa. After 6 days of the fermentation process, strain BKPMF-696 forms 8.28 g of citric acid, while the removal of citric acid is 23.0 g / (dm ³ • day); yield of citric acid from spent sugar 82.8% biomass productivity 15.3 g of citric acid per 1 g of air-dried biomass.

 The known VKRM-501 strain is significantly inferior to the new strain in all respects, especially in the yield of citric acid per unit of air-dry biomass (10.4).

Example 3. To determine the stability of the properties of strains BKPMF-501 and BKPMF-696, activity tests were carried out on stored seed (fungal spores). The activity of the inoculum obtained during the propagation of elite cultures, the activity of inoculum after 6 and 12 months of storage in laboratory conditions at 20 ± 1 ^o C and relative humidity of 75% was checked
The main raw material for the preparation of fermentation media is granulated sugar. The composition of the media is described in example 1. The sugar content in the medium is 150 g / dm ³ ; 8 g of granulated sugar is consumed per flask.

 The results of comparative tests of seed of the two strains are presented in table.2.

 The activity of the initial seed material of the two strains is relatively little different except for the mycelium productivity of strain BKPMF-696, the productivity of mycelium (citric acid yield per 1 g of air-dried mycelium) is 1.5 times higher than that of strain BKPMF-501.

After 6 months of storage, the activity indicators of the strain BKPMF-696 remain at a high level of sugar to citric acid conversion; 90% of citric acid removal from 1 dm ³ / day. is 24.0 g in the compared strain BKPMF-501 within 6 months of storage, the yield of citric acid from sugar decreases from 82% to 70%; I eat citric acid from 21.9 g to 18.7 g.

 Over the next 6 months of storage, seed activity of strain BKPMF-696 does not change activity indicators.

In the compared strain BKPMF-501, there is a further deterioration in sugar conversion after 12 months of storage is 65.25%, the intake of citric acid is reduced to 17.4 g per 1 DM ³ / day.

 Thus, the new strain of Aspergillus niger BKPMF-696 that we select, which forms light-colored conidia, differs significantly from the known strains by the high yield of citric acid from sugar, the stability of the yield of citric acid, and the activity of acid formation during long-term storage.

 The strain is a competitive producer of citric acid in the fermentation of glucose-mineral and sucrose-mineral media.

Claims (1)

 The strain of the fungus Aspergillus niger BKPF 696 producer of citric acid.

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