RU2192460C2 - Strain of fungus aspergillus niger as producer of citric acid - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: method involves the use of selected strain Aspergillus niger VKPM F-809 for production of citric acid under conditions of submerged culturing. Strain shows high acid-producing activity in expanded range of substrates: in medium of sorghum condensed juice, molasses and sucrose-mineral medium. Conversion of sugars of indicated nutrient media to citric acid is 84.1; 62.9; 87.25%, respectively. Strain is the base for development of pliable technology by using different raw species in a single technological process and ensures to decrease the dependence of production from business conditions in raw market. EFFECT: valuable properties of strain. 1 tbl, 3 ex

Description

 The invention relates to the microbiological industry and relates to the selection of a new strain of Aspergillus niger VKPM F-809 fungus, a producer of citric acid under conditions of deep cultivation on any of the nutrient media: medium from condensed sorghum or molasses juice, or beet sugar.

 Known strains of the fungus Aspergillus niger - producers of citric acid on media from condensed sorghum juice / 1 /, on molasses / 2, 3 / and sucrose-mineral media / 4 /.

 A disadvantage of the known strains is their strict specificity for raw materials and cultivation conditions, applicability only to the technology for which they were created, and the lack of universality.

 A known strain of the fungus Aspergillus niger is a producer of citric acid / 5 /, capable of fermenting molasses media under conditions of deep cultivation, as well as media from molasses with the addition of edible sugar and raw sugar, and the introduction of sugar in the fermentation process is a technological method aimed at simplifying the process (exclusion of additional culture feed) and activation of acid formation. The introduction of an additional carbon source in molasses medium containing a complex of growth substances does not characterize the ability of the strain to grow on sugar-mineral media lacking growth stimulants and does not indicate the universality of the strain.

 The present invention is directed to a new strain having the ability to produce citric acid on nutrient media based on both plant materials (condensed sorghum juice, molasses) and refined sugar with a fairly high yield of citric acid.

 The selection of a strain with high acid-forming activity on an extended range of substrates is the basis for creating a flexible technology for the use of various types of raw materials in one technological process and will reduce the dependence of production on market conditions in the commodity market.

 As a prototype, the well-known Aspergillus niger VKPM F-719 strain, a producer of citric acid during fermentation of nutrient media from condensed sorghum juice / 1 /, was selected.

 Tests of the Aspergillus niger VKPM F-719 strain on molasses and sucrose-mineral media showed that the conversion of sugars to citric acid was 42.0 and 55.0%, respectively, that is, the activity of citric acid biosynthesis in the strain on these media is not high enough.

 The initial object for the production of the new strain was cultures of the Aspergillus niger VKPM F-719 fungus stored in the VNIIPAKK Collection.

To obtain a new highly effective strain of Aspergillus niger, stepwise selection was carried out using the mutagenic effect of UV rays at a dose of 3.3 thousand erg / mm ² after preliminary treatment of the conidia of the fungus with the chemical mutagen N-nitrosoguanidine at a concentration of 0.5% with a exposure time of 15 min. The combined effect of mutagens was used in combination with stabilizing selection of selected mutants with the isolation of the most stable spontaneous variants. The selection of options was carried out according to a set of attributes: acid formation activity on various media (condensed sorghum juice, molasses, sugar), stability of morphological and cultural characteristics, and yield of conidia of mushroom cultures.

 The most stable and active mutant strain had a light beige colony of conidia.

 The new breeding strain of Aspergillus niger in the All-Russian collection of industrial microorganisms is assigned a collection number VKPM F-809.

Cultural and morphological properties of the strain Aspergillus niger VKPM F-809. On wort agar after 5 days of cultivation at 32 ^o With the strain forms a colony with a diameter of 9 cm. Colony color is light beige. Aerial mycelium is well developed, conidia is plentiful. The reverse side of the colony is white with slight folding. Conidial heads are rounded in shape, their diameter varies from 50 to 200 microns. Conidial heads with a diameter of 85-200 microns prevail in the center of the colony.

 Apical extensions of conidiophores rounded or slightly elongated with a diameter of 38 • 42 microns. Sterigmas are bilayer. The dimensions of the sterigm of the first layer are 10.8 μm, the second layer is 5.6 μm. The average diameter of conidia is 3.8 microns. The color of conidia is light beige. Conidiophores are straight, colorless, 418-1570 microns long, 11-15 microns wide.

 On Chapek’s environment after 5 days. the cultivation strain forms a colony with a diameter of 4 cm. Colony coloration is light beige. Conid formation is moderate over the entire surface of the colony, with the exception of the asporogenous margin, which is white in color. The reverse side of the colony is white, smooth.

Physiological and biochemical properties. Aerobe. The growth temperature range is 26-36 ^o C. The optimum growth temperature is 32 ^o C. The optimum pH is 6.8-7.2.

 Assimilation of carbon sources: abundant growth on sucrose, D-glucose, fructose, D-galactose; moderate growth on maltose, mannitol, sorbite; poor growth on lactose and starch.

 Assimilation of nitrogen sources: the strain assimilates the nitrogen of organic compounds (proteins, peptones, amino acids) and the nitrogen of mineral compounds (ammonium salts, nitrates).

 Genetic features: prototroph.

Storage method. The strain is stored in the form of dried conidia (5-8% residual moisture) at 18 ^o C.

 The strain Aspergillus niger VKPM F-809 was identified by the definition of fungi of the genus Aspergillus / 6 /.

 The invention is illustrated by the following examples.

 Example 1

For laboratory testing, the culture of the fungus of the selected strain of Aspergillus niger VKPM F-809 is grown in test tubes on wort agar (sugar concentration 7 degrees Balling, agar-agar 20 g / dm ³ , pH 5.8). After 7 days of cultivation at 32 ^o With conidia are removed from the surface of the culture, dried to a residual moisture content of 5-8% and stored at room temperature. Dried conidia are used as seed for evaluating the acid-forming activity of the fungus on various media.

Producer conidia preparation
Conidia of the fungus in an amount of 0.3 g are placed in 100 cm ³ medium of the following composition, g / dm ³ :
Sugar - 50.0
Potassium Dihydrogen Phosphate - 0.16
Ammonium nitrate - 2.5
Magnesium sulfate, heptahydrate - 0.25
A suspension of conidia in the medium is placed on a rocking chair with a speed of (160 ± 5) min ^-1 for 5-6 hours at (32 ± 1) ^o C for swelling and the beginning of germination of conidia.

Fermentation of media based on condensed sorghum juice is carried out in laboratory conditions under deep cultivation of the strain on a shaker with a speed of (160 ± 5) min ^-1 in flasks with a capacity of 750 cm ³ at (32 ± 1) ^o C.

Preparation of culture medium for growing seed mycelium 36 g of concentrated sorghum juice is dissolved in 250 cm ^{3 of} hot water, autoclaved at 0.075 MPa for 30 minutes, cooled to 45-50 ^° C and 7.5 cm ³ 10 wt. % solution of magnesium sulfate heptahydrate and bring the volume to 300 cm ^{3 with} sterile water. The concentration of sugar in the medium is 50 g / DM ³ .

Preparation of fermentation broth
51 g of Concentrated sorghum juice is dissolved in 250 cm ^{3 of} hot water, autoclaved at 0.075 MPa for 30 minutes, cooled to 45-50 ^o C and sterilely introduced 5 cm ³ 10 wt.% Solution of ammonium nitrate, 0.75 cm ³ 10 wt. % solution of magnesium sulfate heptahydrate and bring the volume to 300 cm ^{3 with} sterile water. The concentration of sugar in the medium is 70 g / DM ³ .

Preparation of culture medium for feeding mushroom culture
45 g of Concentrated sorghum juice is dissolved in 150 cm ^{3 of} hot water, autoclaved at 0.075 MPa for 30 minutes, cooled to 45-50 ^{° C.} and 3.4 cm ^{3 of a} 10 wt.% Solution of ammonium nitrate is sterilized. The volume is adjusted to 200 cm ³ . The concentration of sugar in the medium is 100 g / DM ³ .

Cultivation of seed mycelium
Flasks with a capacity of 750 cm ³ containing 50 cm ^{3 of} culture medium inoculate 10 cm ^{3 of a} suspension of conidia. The flasks are placed on a rocking chair with a speed of (160 ± 5) min ^-1 and the fungus culture is grown for 40 hours at (32 ± 1) ^o C.

Fermentation of concentrated sorghum juice
Flasks with a capacity of 750 cm ³ containing 50 cm ^{3 of} culture medium inoculate 5 cm ³ of seed mycelium culture. The flasks are placed on a shaker with a speed of (160 ± 5) min ^-1 and grown at (32 ± 1) ^o C for the first or second day, then the temperature is reduced to 28 ^o C until the end of fermentation. The total growing time is 5 days.

On the second and third days, the fungus culture is fed with 14 cm ^{3 of the} nutrient medium once a day.

 After 5-day fermentation, the fungus culture is inactivated by boiling for 3 minutes and the concentration of organic acids, the amount of citric acid, and its yield from sugar are determined.

 The data are presented in the table.

 As a control, we used the results on the fermentation of media from condensed sorghum juice using the Aspergillus niger VKPM F-719 strain (prototype). Comparative data show that the new strain provides an increase in the conversion of sugars to citric acid to 84.1%, and an increase in the proportion of citric acid in the sum of synthesized acids to 98.4%.

 Example 2. Preparation of seed and preparation of a suspension of conidia is carried out analogously to example 1.

Fermentation is carried out on molasses in laboratory conditions on a shaker with a speed of (160 ± 5) min ^-1 in flasks with a capacity of 750 cm ³ at (32 ± 1) ^o C.

Preparation of culture medium for growing seed mycelium
The experiments are carried out on a molasses sample having the following characteristics: sucrose 46 wt.%, CaO 1.1 wt.%, PH 6.3.

The composition of the medium for growing seed mycelium, g / DM ³ :
Molasses - 65.2
Potassium hexacyanoferrate hydrate - 0.14
Ammonium oxalate hydrate (for the complete precipitation of calcium salts based on CaO) - 1.53
Potassium dihydroorthophosphate hydrate - 0.14
Magnesium Sulfate Hydrate - 0.25
Zinc Sulfate Hydrate - 0.005
Tap water, dm ³ - Up to 1
pH - 6.7
To prepare a nutrient medium, a sample of molasses is dissolved in water heated to 80-100 ^o C in a ratio of 1: 1, the field of which is added ammonium oxalate hydrate and potassium hexacyanoferrate in the amounts indicated above. The volume of the solution is adjusted to the specified water. The required pH value is set using sodium carbonate or sulfuric acid. The solution is sterilized in an autoclave at 0.1 MPa for 30 minutes, cooled to 30-34 ^{° C.} and sterile solutions of potassium dihydroorthophosphate salts and magnesium and zinc sulfates are introduced.

The concentration of sugar in the medium 30 g / DM ³ .

Preparation of fermentation broth
For fermentation prepare a nutrient medium containing 30 g / DM ³ sugar, according to the recipe mentioned above, but without the addition of magnesium sulfate. The amount of ammonium oxalate hydrate is reduced by 2 times.

Preparation of culture medium for gravy
The composition of the medium, g / DM ³ :
Molasses - 543.0
Potassium hexacyanoferrate hydrate - 2.58
pH 6.3
The concentration of sugar in the medium, g / dm - 250
Cultivation of seed mycelium
Flasks with a capacity of 750 cm ³ containing 50 cm ^{3 of} culture medium are inoculated with a suspension of conidia in an amount of 10 cm ³ . The flasks are placed on a rocking chair with a speed of (160 ± 5) min ^-1 and the fungus culture is grown for 24 hours at (32 ± 1) (C.

Fermentation of molasses
The medium prepared for fermentation is poured into 50 cm ³ into flasks with a capacity of 750 cm ³ and contribute 10 cm ^{3 of} seed mycelium. The flasks are placed on a rocking chair with a speed of (160 ± 5) min ^-1 and a mushroom culture is grown for 5 days. In the fermentation process, after 24 hours of cultivation, the fungus culture is fed in two doses during the day and at least 5 hours apart.

In one step, 10 cm ^{3 of} molasses solution are added to each flask. The total volume of the feed solution is (20 ± 1) cm ³ .

 After 5-day fermentation, the fungal culture is inactivated by boiling for 3 minutes and the concentration of organic acids, the amount of citric acid and its yield from sugar are determined.

 The data are presented in the table.

 As a control, we used the results of fermentation of molasses media using a strain of Aspergillus niger VKPM F-719 (prototype). Comparative data show that, as a result of the selection of a new strain, the fermentation process increased significantly: the conversion of sugar to citric acid was 62.9%, the mass fraction of citric acid in the total acid increased to 88.0%.

 Example 3. The preparation of seed and the preparation of a suspension of conidia is carried out analogously to example 1.

Fermentation is carried out on sucrose-mineral media in laboratory conditions on a shaker with a speed of (160 ± 5) min ^-1 in flasks with a capacity of 750 cm ³ at (32 ± 1) ^o C.

Preparation of culture medium for growing seed mycelium
For the cultivation of seed mycelium, the following composition is used, g / dm ³ :
Sugar - 50.0
Beet molasses - 17.0
Ammonium nitrate - 2.5
Potassium dihydroorthophosphate hydrate - 0.16
Magnesium Sulfate Hydrate - 0.25
Tap water, dm ³ - Up to 1
the pH of the finished solution is 5.0-5.5
A sugar solution with molasses is sterilized in an autoclave at 0.05 MPa for 30 minutes. Molasses is added to the seed medium to stimulate the growth of the fungus seed mycelium. In cooled to 32 ^o With sugar solution make sterile solutions of salts; the finished nutrient medium is poured into flasks of 60 cm ³ in compliance with sterility conditions.

Preparation of fermentation broth
For fermentation using a nutrient medium of the same composition as for the preparation of seed mycelium, but with an increase in sugar content up to 150 g / dm ³ and the exclusion of molasses. The medium prepared for fermentation is poured into 50 cm ³ .

Cultivation of seed mycelium
Flasks with a capacity of 750 cm ³ containing 60 cm ^{3 of} culture medium are inoculated with a suspension of conidia in an amount of 10 cm ³ . The flasks are placed on a rocking chair with a number of revolutions (160 ± 5) min ^-1 and a mushroom culture is grown for 48 hours.

Fermentation of sucrose-mineral media
Nutrient medium for fermentation is seeded with 10 cm ^{3 of} seed fungus mycelium. The flasks are placed on a rocking chair and a mushroom culture is grown for 5 days. at (32 ± 1) ^o C.

 Culture recharge is not performed.

 After 5-day fermentation, the fungal culture is inactivated by boiling for 3 minutes and the concentration of organic acids, the amount of citric acid and its yield from sugar are determined.

 The data are presented in the table.

 As a control, we used the results of fermentation of sucrose-mineral media using the Aspergillus niger VKPM F-719 strain (prototype).

 A comparison of the fermentation results shows that the selected strain of Aspergillus niger VKPM F-809 significantly exceeds the prototype in all process indicators: the conversion of sugar to citric acid is 87.25%, the mass fraction of citric acid is 100%.

 Thus, as a result of breeding, the strain Aspergillus niger VKPM F-809 was obtained - a producer of citric acid, which has the versatility that ferments nutrient media during deep cultivation: condensed sorghum juice, molasses, and sugar with high rates.

Sources of information
1. RF patent 2088658, IPC 6 C 12 N 1/14, 1997.

 2. RF patent 1315472, MKI 4 C 12 N 1/14, 1987.

 3. RF patent 975799, MKI 3 C 12 N 15/00, 1982.

 4. USSR patent 1811697, IPC 5 C 12 N 1/14, 1993.

 5. RF patent 2088665, IPC 6 C 12 P 7/48, 1997.

 6. Bilay V.I., Koval E.Z. Aspergillus. - Kiev: Naukova Dumka, 1988 .-- 204 p.

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-809 is a producer of citric acid.

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