RU2089615C1 - Fungus strain aspergillus niger - a producer of citric acid - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to the new strain of Aspergillus niger VKPM F-681 - a producer of citric acid. Strain is obtained by selection and has black conidia, diameter - 3.4-4.6 mcm. Strain produces 11.8-18.7 g citric acid/1 g air-dried mycelium. EFFECT: high yield of biomass and citric acid. 1 tbl

Description

 The invention relates to the microbiological industry and the selection of Aspergillus niger, a producer of citric acid for the deep fermentation of media: sugar-mineral and starch hydrolysis containing various amounts of glucose.

 Known natural strain of the fungus Asp. niger 82 (D) producer of citric acid for the fermentation of sucrose-mineral media. After 10 days of fermentation, the strain forms 50-60% of citric acid from spent sugar (1). When cultivated on wort-agar, strain 82 (D) abundantly forms black-stained conidia 4.3 microns in size.

 Known producer of citric acid Asp. niger S-59 (from the CCF 1600 collection of microorganisms) for deep cultivation on a nutrient medium containing 15% sucrose. After 7 days of fermentation, the yield of citric acid reaches 70 75% of the added sugar (2).

 A disadvantage of the known strains 82 (D) and S-59 is the relatively low productivity during prolonged fermentation.

 The prototype of the invention can serve as a known strain of Asp. niger VKPMF-501, designed for deep cultivation on sucrose-mineral media, forming citric acid with a sugar yield of 82.5%. The strain has a beige color of conidia (3).

 The disadvantage of the strain VKPMF-501 is its powerful growth, which requires a significant expenditure of sugar on the formation of biomass to the detriment of the resulting citric acid. Due to the abundant growth, the yield of citric acid from spent sugar in cultures of strain VKPMF-501 is not high enough.

The technical result of the invention to obtain a new strain of Asp. niger, which has a higher productivity for the formation of citric acid during fermentation by a deep method of culture media in which the carbon source is sucrose or a mixture of sugars with a glucose content of 15 to 75%
The initial object for obtaining a new strain by selection was a black-colored version of Asp. niger 163/9 from the collection of VNIIPAKK. A typical form 163/9, when grown on a sucrose-mineral medium, produces 4.5 g of citric acid per flask; the yield of citric acid from sugar is -56%
To obtain a new highly productive strain Asp. niger carried out step selection, consisting of five stages. To induce and select mutants, the initial cultures were exposed to ultraviolet rays. To stabilize the properties of selected mutants, the selection of stable spontaneous variants was carried out. The basis for the selection of active mutants and spontaneous variants were the results of fermentation of sucrose-mineral media and nutrient solutions prepared from caramel molasses and starch hydrolyzate; at the same time, the stability of morphological and cultural properties of selected strains was investigated.

 The new breeding strain of Aspergillus niger in the All-Russian collection of industrial microorganisms is assigned a collection number VKPMF-681.

CULTURAL-MORPHOLOGICAL PROPERTIES OF THE NEW STRAIN VKPMF-681 Aspergillus niger
On the 5th day of growth on wort agar, a giant colony with a diameter of 90 mm. The colony is black-painted. In a radius of 10 mm, the conidiocarrier is plentiful. On the rest of the colony, conidial heads are formed in groups, not very densely. The "sterile" edge of the colony with immature conidial heads is about 5 mm. From the bottom of the colony in its center is a dense, peripheral part of the colony has a thin mycelium.

 After 10 days of cultivation on wort agar, the culture is characterized by copious maturation of conidia. Conidial heads from 54x54 microns to 180x234 microns in diameter, an average of 119 microns. Swelling of conidiophores 34-39 microns spherical or longitudinally elongated. Sterigmas are two-layer. The length of the sterigm of the first layer is 9x2.3 11.5x4.6 microns; the sterigma length of the second layer is 4.6 μm. Conidia are round, their diameter is 3.4-4.6 microns, an average of 3.99 microns. The shell is gray, has many spikes. Conidiophores are straight, their length is 240-1000 microns; average length 450 microns. Cross section of conidiophores 19-34 microns; an average of 29 microns.

 Conidiophores unpainted, a dark brown pigment accumulates in the upper part of the conidiophores adjacent to the vesicle.

 Compared with the parent strain 82 and the original 163/9 strain, the new strain has a lower morphological structure (length of conidiophores, bloating of conidiophores, second layer sterigma, conidia): dark gray color of conidia differs from the prototype of the beige strain VKPM-501.

 Strain Asp. niger VKPMF-681 was identified by the Qualifier of fungi of the genus Aspergillus (4).

PHYSIOLOGICAL-BIOCHEMICAL PROPERTIES Aspergillus Niger VKPMF-581
Aerob, cultivation temperature 28-32 ^o C, pH 5.5 to 7.5.

 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.

Storage 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), protected from direct sunlight.

GENETIC FEATURE OF THE STRAIN-PROTOTROPH
The invention is illustrated by the following specific examples of the use of strain VKPMF-681.

Example 1. For laboratory testing, seed is grown in test tubes on wort agar; for semi-production and 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 elite culture of the fungus and incubated in a thermostat at 32 ^o C for 7 days. The relative humidity in the thermostat gradually decreases: the first four days from 80 to 60% the last three days from 60 to 40%. For complete ripening, the fungus culture is kept at room temperature for 2 days. After 9-10 days, conidia are removed from the surface of the mushroom films 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 by the deep method on a rocker AVU-50r with a number of swings of 160-180 per min. The temperature in the rocking room is 32 ^o C.

 Culture media is prepared in 750 ml flasks; the volume of medium is 50 ml.

A fungal culture grown on wort agar for 10 days or air-dried conidia (10 ⁷ conidia / cm ³ medium), the medium is inoculated and inoculated mycelium is grown for 2 days. The temperature in the heat chamber is 30-32 ^o C.

 The main raw material for the preparation of media in this example is granulated sugar.

 The composition of the nutrient medium (g / l): sugar 50, beet molasses 17, ammonium nitrate 2.5, seven-water magnesium sulfate 0.25, potassium dihydrogen phosphate 0.16, tap water up to 1 liter, pH 5.0 5.5.

 10 ml of grown mycelia are seeded with 50 ml of fermentation medium. The composition of the fermentation medium differs from the composition of the nutrient medium with an increased sugar content of up to 150 g / l. In the fermentation process, culture is not fed. In total, 8.0 g of sugar is spent on growing seed mycelium and on fermentation. The media is sterilized in an autoclave by fluid steam for 15 minutes and at 0.5 atm 30 minutes. The accumulation of citric acid is determined using the methods described in the Instructions for the biochemical and chemical control of the production of edible citric acid, L. 1972.

After 5 days of fermentation, 7.18 g of acids are formed from 8 g of sugar, including 99.2% of citric acid and 0.8% of gluconic acid; I will remove citric acid with dm ³ / day 23.7 g, the yield of citric acid from the introduced sugar 89.0%
Example 2. Inoculum of a new strain is procured according to the method described in example 1, the main raw material for the preparation of fermentation media in this example is caramel syrup containing glucose, maltose, dextrins (the amount of fermentable sugars is 69.8% glucose 15%). From conidia of the fungus for 2 days in depth on a rocking chair AVU-50r, seed mycelium is grown on a nutrient medium of the composition, g / l: caramel syrup 71.6, ammonium nitrate 2.5, magnesium sulfate seven-water 0.25, potassium dihydrogen phosphate 0, 16, tap water up to 1 liter.

10 ml of grown mycelia are seeded with 50 ml of fermentation medium prepared in 750 ml flasks. The composition of the fermentation medium (g / l): caramel syrup (glucose content in the sugar mixture 15%) 171.9, ammonium nitrate 1.6, magnesium sulfate seven-water 0.25, potassium dihydrogen phosphate 0.16, zinc sulfate 0.005. Tap water up to 1 liter. The media is sterilized for 15 minutes with fluid steam and 20 minutes at 0.5 atm. 6.5 g of sugar is spent on cultivating seed mycelium and fermentation. After 5 days of fermentation, 6.01 g of citric acid accumulates in the medium. The yield of citric acid from sugar is 92.5%. Removal of citric acid from 1 dm ³ / day 20.0 g.

Example 3. The seed of the new strain is harvested according to the method described in example 1. Cultivation is carried out on a rocking chair in the same conditions. The main raw material for the preparation of fermentation media in this example is caramel syrup. Inoculum mycelium is grown on the medium of the composition given in example 2. Growing mycelium in an amount of 10 ml is seeded with 50 ml of fermentation medium. The composition of the fermentation medium differs from that shown in Example 2 by the increased sugar content: sugar consumption per cycle of 7.5 g. After 5 days, fermentation in the medium accumulates 6.9 g of acid. In the sum of acids, citric acid is 99.39% gluconic 0.61%. Removal of citric acid in terms of 1 dm ³ / day 22.9 g, citric acid yield from sugar 91.5%
Example 4. The seed material of a new strain is procured according to the method described in example 1. The raw material for the preparation of fermentation media in example 3 is a starch hydrolyzate, the glucose content of 74.9%. The fungal seed mycelium is grown on a rocking chair AVU-50r for 2 days on a medium composition ( g / l): starch hydrolyzate 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 liter. Seed mycelium in an amount of 10 ml is introduced into the fermentation medium of the composition (g / l): 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 a day, a 25% solution of starch hydrolyzate in an amount of 24 ml is added in two doses to the fermentation medium. The sugar consumption per flask is 10 g. The nutrient and fermentation media are sterilized for 15 minutes with fluid steam and 20 minutes at 0.5 atm. The air temperature in the heat chamber is 32 ^o C. After 7 days of fermentation, 8.4 g of citric acid accumulate in the fermentation medium, the yield of citric acid from sugar is 84%
From the above examples and the attached table it can be seen that the new strain VKPMF-681 grown on sucrose-mineral media significantly exceeds the known Asp strains. niger 82 (D), S-59 and VKPMF-501 for the extraction of citric acid per unit volume of medium per day and the yield of citric acid from spent sugar. When cultured on fermentation media prepared from starch hydrolysis products containing glucose, the new strain significantly exceeds the prototype strain VKPMF-501 in the removal and release of sugar from citric acid.

 The big advantage of the new strain is limited growth and high productivity of mycelium, reaching 18.7 g of citric acid per 1 g of air-dried mycelium for 5 days of fermentation; the strain does not form oxalic acid; the mass fraction of citric acid in the sum of acids is 94-99%; the yield of citric acid from sugar spent on the cycle is 84-92.5% (see table).

 The new strain is quite versatile and is proposed for fermentation of sucrose-mineral media and media obtained by hydrolysis of starch containing from 15 to 75% glucose.

INFORMATION SOURCES
Zhuravsky G.I. Deep way to produce citric acid. - Microbiology, 1955, vol. XXIV. issue 3, p. 323-340.

 Patent 202709 Czechoslovakia, cl. C 12 P 7/48. Zpusob vyroby kyseliny citronove submersnum zpusobem zkvasovanum cukernych roztoku pliseni Asp. niger. - Zerpold S. (Czechoslovakia), Musilkova M. (Czechoslovakia), Fencil Z. (Czechoslovakia), Ujcova E. (Czechoslovakia), publ. 05/30/80.

 Mushroom strain Asp. niger VKPMF-501 producer of citric acid. - V.M. Golubtsova (USSR); V.P. Ermakova (USSR); E.S. Mints (USSR); T.A. Nikiforova (USSR); A.V. Galkin (USSR); V.M. Finko (USSR); V.N.Zhdanova (USSR).

 Bilay V.I. Koval E.Z. The genus Asperilli - Kiev. Naukova Dumka, 1988.

Claims (1)

 The strain of the fungus Aspergillus niger VKPM F-681 producer of citric acid.

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