RU2088658C1 - Fungus strain aspergillus niger - a producer of citric acid - Google Patents

Abstract

FIELD: biotechnology, microbiological industry. SUBSTANCE: invention proposes the new strain of fungus used for citric acid production under conditions of submerged culturing. Breeding strain Aspergillus niger VKPM F-719 shows high activity with respect to citric acid synthesis at submerged culturing on media containing the condensed sorghum juice. Output of citric acid is 18.0-18.7 g/dm³ x 24 h. Conversion of sugars from the nutrient medium is 79.2-80.1%. EFFECT: increased yield of citric acid. 1 tbl

Description

 The invention relates to the microbiological industry and relates to the selection of a new strain of Aspergillus niger fungus producer of citric acid under conditions of deep cultivation on media from condensed sorghum juice.

 As a prototype, a well-known industrial strain of the fungus Aspergillus niger VKPM F-326, a producer of citric acid, selected for fermentation of molasses media, was selected. The strain is strictly specific to the conditions of fermentation and its use for a new type of raw material is impractical because it does not provide high rates of the fermentation process (table).

 The invention is directed to a new strain having a high activity of citric acid biosynthesis during fermentation of media from condensed sorghum juice.

 The initial object for obtaining a new strain was light-colored cultures of Aspergillus niger, preserved in the collection of VNIIPAKK.

 To obtain a new highly productive strain of Aspergillus niger, stepwise selection consisting of four stages was carried out. To induce mutants, the initial cultures were exposed to UV rays, the obtained mutants were stabilized by selection of stable spontaneous variants.

 The most stable and active mutant strain had a dark beige color of conidia.

When cultivated on media from condensed sorghum juice in deep conditions for 5-6 days of fermentation, the new strain provides a yield of citric acid from the sugar of the nutrient medium of 80.1%, which significantly exceeds the strain selected for molasses media (table). In addition, the strain allows you to ferment nutrient media from condensed sorghum juice with a sugar concentration increased to 137 g / dm ³ with high rates: the intake of citric acid was 18.0 g / dm ³ • days, the conversion of sugars to citric acid 79.2 % which also indicates the significant advantages of the new strain compared with the strain of the prototype.

 The new breeding strain of Aspergillus niger in the All-Russian collection of industrial microorganisms is assigned a collection number VKPM F - 719.

 Cultural and morphological properties of the strain Aspergillus niger VKPM F-719.

On wort agar after 5 days of cultivation at 32 ^o C forms colonies with a diameter of 9.4 cm. Colony color is dark beige. Aerial mycelium is well developed, abundant conidia. In the center of the colony, the conidial heads are larger and form a slight elevation. The reverse side of the colony is smooth, white. Conidial heads are round in shape, their diameter from 62 to 128 microns. Swelling of conidiophores rounded or slightly elongated, with a diameter of 30-34 microns. Sterigmas are bilayer. The sizes of sterigm of order I are from 9.3 to 14.6 microns, of order II6.65 ± 0.15 microns. The average diameter of conidia is 3.9 microns. Conidiophores are straight, colorless, from 318 to 1290 microns long, conidiophores 950 microns in length prevail.

 On Chapek’s medium, after 5 days of cultivation, colonies with a diameter of 6.5 cm are formed. Colony coloration is yellowish-beige. Aerial mycelium is well developed, abundant conidia. Along the edge of the colony in a radius of 0.6 cm, the colony has a white color, due to immature conidial heads. There is a small number of immature conidial heads in the center of the colony and over its entire surface. The substrate mycelium is light, has a small radial folding.

Physiological and biochemical properties
Aerobe. The temperature range of growth is 28-34 ^o C. The optimum growth temperature is 32 ^o C. The optimum pH is 6.8 to 7.2.

 Assimilation of carbon sources: abundant growth on sucrose, D-glucose, fructose, D-galactose, maltose, mannitol, sorbite: lactose and starch are not assimilated.

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

 Storage method.

The strain is stored in the form of dried conidia (5-10% residual moisture). Storage temperature 16-18 ^o C.

 Genetic features: prototroph.

 The strain Aspergillus niger VKPM F-719 was identified by the determinant of fungi of the genus Aspergillus.

Example 1. For laboratory testing, the seed of the selected strain of Aspergillus niger VKPM F-719 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 ^° C, conidia are removed from the surface of the medium, dried to a residual moisture content of 5-8% and stored at room temperature.

 Preparation of conidia producer.

Conidia of the fungus in an amount of 0.3 g per 100 cm ^{3 is} placed in the medium for soaking the following composition, g / DM ³ :
Sugar sand 50
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 min ^-1 and incubated for 5-6 hours at 32 ^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 for 30 minutes at 0.075 MPa, cooled to 45-50 ^o C and 7.5 cm ^{3 of a} 10% solution of ammonium nitrate is sterilized, 0.75 cm ³ 10% solution of magnesium sulfate heptahydrate and adjusted to 300 cm ^{3 with} sterile water. The concentration of sugar in the medium is 50 g / DM ³ .

 Preparation of a nutrient medium for fermentation.

51 g of concentrated sorghum juice is dissolved in 250 cm ^{3 of} hot water, autoclaved for 30 minutes at 0.075 MPa for 30 minutes, cooled to 45-50 ^° C and 5 cm ^{3 of a} 10% solution of ammonium nitrate are sterilized, 0.75 cm ³ 10% 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 .

Preparing a culture medium to fuel the fungus culture. 48 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% 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 min ^-1 and grow the culture of the fungus for 40 hours at a temperature of 32 ^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 min ^-1 and grown at 32 ^o C for the first or second days, 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 day, the fungus culture is fed with 14 cm ^{3 of the} nutrient medium once a day.

 After 5-day fermentation, the fungal culture is inactivated by boiling for 3 min and the amount of citric acid, its yield from sugar, and citric acid are removed from a unit volume of medium per day of the fermentation process.

 The data are presented in the table.

 Example 2. The preparation of conidia producer, preparation of a nutrient medium for growing seed mycelium, fermentation, growing seed mycelium, fermentation is carried out analogously to example 1.

 Preparing a culture medium to fuel the fungus culture.

76.9 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% solution of ammonium nitrate is sterilized and the volume is adjusted to 200 cm ³ . The concentration of sugar in the medium is 160 g / DM ³ .

 Mushroom culture feed.

On the second or third day, 12.5 cm ^{3 of} nutrient medium are sterilely introduced into the flasks.

 Further operations are similar to example 1.

 The data are presented in the table.

 Example 3. The experimental conditions are similar to example 1.

 As a producer of citric acid, the known Aspergillus niger VKPM F 326 strain is used.

 The data are presented in the table.

The data presented in the table show that the proposed strain on a medium of sorghum juice allows to achieve a removal of citric acid of 18.7 g / dm ³ • days, the conversion of sugars to citric acid 80.1 at the same time with strain VKPM F-326 under the same conditions of fermentation, these indicators are significantly inferior: the removal of citric acid 16.32 g / dm ³ • days, the conversion of sugars to citric acid 69.5
Thus, the selected strain Aspegillus niger VKPM F -719 is an active acid-forming agent on media from condensed sorghum juice and provides high rates during fermentation of culture media.

Sources of information
1. Application N 94041280/13 PF, MKI 5, C 12 P 7/48, 1994.

 2. Pat. 1345472 USSR, class C 12 N 1/14, 1987.

 3. V.I. Bilai, E.Z. Koval. Asperigillus. Kiev, Naukova Dumka, 1988, p. 99.

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-719 producer of citric acid.

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