SU975799A1 - Strain of fungus aspergillus niger l-4 producer of citric acid - Google Patents

Description

This invention relates to microbiological clarity and relates to a new strain of the fungus Asperglllus nlfjer, producing citric acid. The strains of the fungi Azregdälus Nine P-1 and L-106 are known, used to produce citric acid by the f11 and 2j method. . The duration of conidia germination in these strains is 9 hours. During this time, concomitant microoil develops, inhibiting the acid production of the main producer. A known strain of Asperrjillus niger L-1, used as a producer of citric acid in production conditions. However, this strain is applicable only to molasses with a low initial sugar concentration. The purpose of the invention is to obtain a strain with a high activity of citric acid formation during the fermentation of molasses solutions with a high initial concentration. was obtained as a result of the selection of strain L-1 by the influence of the combined effect of chemical and mutagenic factors: diazoacetylbutane (DAV) and UV rays, diethyl sulfate (DES) and PP rays, cycloAosfan (CA) and UV rays. After each treatment method, acid formation was studied in 100 mutants. The combined effect of the DAA solution for 3 hours and UV irradiation at a dose of 3.3 thousand erg / mm turned out to be most effective. Thus, mutant N, hereinafter referred to as strain L- (Leningradsky-), was selected. A new strain abundantly forms a beige-colored conidia, which is sown with seed material in the production of citric acid; conidia germinate after 6–7 hours. Ptamn is more resistant to bacterial flora associated with citric acid production, compared with production strain L-1. Ptamma L-is an active producer of citric acid both under surface cultivation and under deep conditions during the fermentation of molasses media of high concentration, while citric acid in the composition of synthesized acids is 75-90%. Strain L is an active acid former under surface and deep cultivation on media prepared from molasses. Under cultivation in deep conditions on concentrated molasses media, the strain forms 9, kg of citric acid per 1 m per day, the flow rate of 6% molasses per 1 ton of citric acid is 2959 kg. Under surface cultivation on molasses media, the strain L = about 1.02 kg of citric acid from 1 day, the consumption of 6% molasses per 1 ton of citric acid is 3173 kg. Whether the yield of young acid from sugar is (when using strain L-) is 75% in deep conditions and 69 when cultivated by the surface method. The formation of citric acid in strain L-C has been studied under various modes of process control: in laboratory experiments under deep growing conditions on molasses media with a sugar content of 10 and 13% (Table 1); in laboratory experiments in deep conditions on molasses media diluted up to 3% in sugar content according to plant technology (Table 2); in the production cycle in the plant of deep-seated fertilization of the Leningrad plant for the production of citric acid on melamine media with an initial sugar concentration of 10% (table. in laboratory conditions with surface growing on glasses on molasses media, on sugar. Test results of the proposed strain L compared to with known L-1 are summarized in Table 1 of Table 3. It follows from the tables that strain L is a more active kialothobial in all the modes tested as compared with production strains. Productivity of a new strain and on concentrated media containing 9–15% sugar, increases by 11–29% compared with the activity of the production strain L-1 under the same conditions. Ptamm of the fungus Asperglllus niger L is deposited in the All-Russia Research Institute of Genetics under the number F-171. The following characteristic: Morphological and physiological characteristics. Mor (the juridical properties are studied from five daily cultures grown on the апapek medium. Conidial heads of a round YOR, their diameter is μm, the average head is 90.1 μm. Swelling of a conidiophore (bubble) of a globular shape with a diameter of 20 x 29 to 8–60 µm, average diameter (x 3 µm µm. Sterigma bilayer: pair sterigm length of 6–32 µm, average 13.3 µm. Sterigm of the second order length 5-10 µm, average 6.5 µm. Conidia are round with a thick spinous cream shell. The average diameter of conidia is 1 µm. Conidiophores are direct, colorless, the length of conidiophores is tlS1265 µm, width is 9-20 µm. Antagonistic properties of strain L-4. development of E.coti, Bacitlus subtitls, flacijtus mesentericus bacteria associated with citric acid production, and is a stronger antagonist in relation to this microflora in comparison with the production strain L-1. Cultural and (biochemical characteristics. Wort agar. Colony is fast-growing, reaching an average of 8.6 cm in diameter on the 5th day. Color of the colony is light -bezheva. Aerial mycelium is highly developed. In the center of the colony the conidial heads are located tightly. The conidial heads are smaller from the center to the edge of the colony. Along the edge of the colony within a radius of 0.6, cm there is a light zone with immature white conidial heads tightly pressed to the substatic mycelium. The reverse side of the colony is smooth white. Wednesday Capek. Colonies with a diameter of 6.1 cm. The colony is beige. In the center of the colony, the conidial heads are large, tightly arranged, forming an elevation. The size of the conidial heads is reduced to the edge of the colony. The growing edge within a radius of 0.5 cm covers the immature small conidial heads. The reverse side of the columns is smooth white. In contrast to the production strain L-1, the new strain LF forms smaller colonies. Mor (1 logically differs from production strain 4a by a slightly smaller size of conidial heads and longer first-order sterigmas. The third strain is a stronger antagonist (compared to baseline) with respect to the accompanying bacterial flora. Example 1. Growing an acid-forming micelle and biosynthesis of acid was carried out in a laboratory on an AVU-50p shaker with a number of swings of 160 per minute in flasks with a capacity of 700 cm at. The molasses solution P1 was rolled over according to the technological design of Io for the production of citric lemon Y1 LOTY1970). The experiments were carried out on molasses of the Leningrad sugar plant of the 1977 billet. Molasses had the following characteristics: sucrose CaO 0, solids 75. Quality is 63.5% (pH, 35. Unlike industrial cultivation, nietz was grown on molasses media ” containing 5 "sugar and fermentation in 10% sugar-rich molasses media. Composition of growth media for spores g / l: Molasses 10 Orocyanide of potassium 0,25 Carbohydrate sodium anhydrous 0.32 1 | ammonium sour ammonium 2, 01 Odosubstituted () osmium potassium 0 , 16 Zinc sulfate, water 0.005 Sulf magnesium magnesium 0.25 The following brothfrom medium, g / l: Molasses 208 () potassium erocyanide 0.5 Sodium carbonate, anhydrous O, 65 Single potassium phosphate 0.16 water zinc sulfate 0, 005 For preparing a spore suspension 0.1 g Aspergillus nlfier spores were soaked in a 10 ml molasses solution for 1 hour at. Prepared 5% molasses solution for growing were poured into 50 ml flasks and seeded with 10 ml of a Spore suspension. In this medium, spores were grown 2 (h. Podroshchenny mycelium served as a seed for seeding fermentation media. Fermenting molasses media with a sugar content of 10% were poured into 50 ml flasks and 10 ml of susceptible mycelium were added to them. The whole fermentation process proceeded on the original fermentation the solution without additional fractional topping of the nutrient medium and ended after 5 days (Table 1). The control variants were experiments in which the fermentation process was carried out under the same conditions, but the production strain L-1 served as the causative agent of the fermentation ( 1). Tests have shown that on molasses media with a sugar content of 10% j, the selection strain L-increases the removal of citric acid by 12.9 "and gives the economy of the main raw material of molasses by 11% compared with the original strain L-1 cultivated under the same conditions. Example 2. The growth of mycelium was carried out on molasses media containing 5% sugar, fermentation was carried out using molasses media with a sugar content of T3%. The molasses medium prepared according to Example 1 was used for growth. For fermentation they prepared 13% - sugar content molasses medium containing, g / l: Molasses270 Potassium ferrocyanide 0.65 Sodium carbonate, anhydrous 0.65, 7, 9 Single potassium phosphate 0.16 Water zinc sulfate0.005 The rest of the process was carried out according to the technology described in example 1 The process of broshsen on (sugar) molasses solution (Table. 1} strain L-increased removal of citric acid on and off the consumption of the main raw molasses Ia t SiH compared with strain L-1, bought “) m under the same conditions. Example 3. The process of citrate fermentation was carried out in deep conditions (not technology currently adopted at citric acid plants). Dp spraying spores were prepared according to sugar molasses grow. thief containing, g / l5 Molasses62 Potassium ferrocyanide 0.15 Sodium carbonate, anhydrous 0.15 G1avele to acid ammonium 1, 2 One -substituted potassium phosphate 0.16 Zinc sulphate aq. 005 Water magnesium sulphate0.25 Fermentation was prepared for sugar fermentation fermentation (composition growth medium), but without the addition of magnesium sulphate and ammonium oxalate. The composition of the medium for gravy ,, g / l: molasses 521.0; potassium ferrocyanide 1.25. Growing mycelium was carried out in one stage on the (sugar) molasses solution. For fermentation, a molasses solution was prepared (for sugar). After 2 m after seeding of the brood solutions with spawned mycelium, additional gravy of molasses solution with a content (L heat of 25%) was started. Gravy was performed 2 times by 18.8 MP in k hours (Tr. 2). When conducting the process on dilute molasses, X media the production strain of strain L-1 in the composition of the synthesized acids citric acid was Bk, 6%, in the new strain L-90, liquefied acid in strain L-1 - 9, fi g / cm per day, in strain L- 4 - 11.96 g / cm per day. Save the main raw material of molasses in strain nk is 9.1. Example t. Leveling the bottom of the former "1 The goals of the L-strain and citric acid synthesis were carried out by a deep method on a concentrated medium under production conditions in the deep fermentation workshop of the Leningrad citric acid plant. A mixture of conidia, seed sowing, fermentation, and topping up was made from one and that he molasses with the mode of preparation used for it. When conducting the process using strain Lk on a concentrated medium, the growing was carried out in 5 m fermenters in a 3 m molasses medium with a sugar content of 5%. After 2 h, the sprouting mycelium in a volume of 3 m was transferred to a fermentation fermenter containing 30 m of a 10% sucrose molasses solution. For 3 days, the volume of the solution was maintained at 2 m, with sterile water. After three days, the ball began to add molasses solution. Gravy was carried out after 1.5 h in 1000 l. With gravy made 1I8 kg of sugar. With such a process, the composition of the synthesized acids formed 77.6% citric acid. The volume of citric acid from 1 m per day was 9.31 kg, the yield of citric acid from sugar was 71.1. In parallel, a control cycle was conducted in which the producer was production strain L-1. At the same time, the citric acid fermentation process was carried out according to a known technology, growing was carried out in 5 m Liter on a sugar and molasses medium for 2k hours, then 3 m of a grown mycelium was transferred to a fermentation fermenter containing 27 m Zol-o molasses. After 2k hours, a molasses solution was added to a 25% sugar mixture. Introduced in 1 m solution every 1.5 m. With dolivam made kg of sugar. The cycle duration was 6.5 days. With this introduction of process 9, 80.3% citric acid was formed in the composition of the synthesized acids. Citric acid from 1 m per day was 8.6 kg, the yield of citric acid from sugar was 78.9. Testing under production conditions showed that the L-strain is an active producer of citric acid on concentrated molasses media, increasing the removal of citric acid by 9.2% compared with the L-1 strain, in accordance with the plant technology. Example 5 The test was carried out in laboratory conditions by the permanent permanent method. The composition of the molasses medium is the following g / l: Beet molasses (sugar in molasses .7%) 360.0 Anhydrous sodium carbonate0,8 Potassium hydrocyanide 0.81 Potassium phosphate monosodium hydroxide 0.111 black sulfate 0.005 RI 7.18 Molasses medium in the amount of 500 ml ( a layer thickness of 12 cm was poured into chemical glasses from the bottom of 0.41 dm of the Conidia strain L-sown on the surface of the medium. Duration podraschivani 8 days, temperature. In parallel, the production of the first strain P-1 was grown at the optimum mode for it. The molasses medium recipe for strain P-1 is as follows, g / L Beet molasses (sugar in molasses, 7) 330 Anhydrous sodium carbonate, 0, 9 Potassium ferrocyanide 1.3 Potassium phosphate monosubstituted 0.11 Zinc sulfate 0.005 pI 7.2 At the end of the experiment strain L received 78.16 g of citric acid on average per cup. Removal of moloic acid in terms of 1 m per day Composition 1L g, output from caixapa 69%. 9 On average, production of strain P-1 produced 5.12 g of citric acid per cup. The content of citric acid from 1 m per day was 1650 g, the yield of ligunic acid from gahara is 69%. Thus, the proposed Aspergillus nlfjer L-strain is a highly active acid forming agent as compared to the L-1, P-1. and L-106 weights that are used when cultured on media from the molasses in the deep and superficial way. Under deep conditions on diluted molasses media (according to the applied technology), strain P – k provides an increase in the volume of citric acid by 21.0 and saves molasses compared to strain L – 1, we use w in the production according to the same technology by 18, one%. Under deep conditions on concentrated media from molasses containing 9–13% sugar, strain L-4 provides an increase in the volume of citric acid as compared to strain L-: 1, spliced under the same conditions by 12, and the economy of molasses is by 11 ,, 1% In addition, the use of strain L-on concentrated media significantly reduces the complexity of the technological process: the number of cooking slurry molasses solutions is reduced, less capacity is required for the preparation and storage of solutions; the number of gravy is reduced by half and the need to hold them during the period when the producer is particularly susceptible to infection. On surface cultivation on media from molasses containing sugar, the Pg-k strain increases the removal of gyc citric acid compared to strains P-1 by 23.7%. The advantage of strain L-consists in the fact that xygidia of this strain germinate after 7 hours, and in strains P-1 and L-106 - after 9-10 hours. The use of a new strain L-makes it possible to proceed to the fermentation of molasses media with an elevated by the addition of sugar in the initial solutions used for fermentation, which will lead to the intensification of citric acid fermentation in a killed manner.

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invention claims 1. Copyright S1L1 in the USSR

The strain of the fungus AsperglHus nlger L- -N360677. cl. П 12 Р 7АП, 19/5. (collections of the Central Museum of Industry Mouuur °° and ° Institute;,., USSR author's certificate

spm ° 778260. class. C 12 N 15/00

CMPM F-171) - producer of citric acid 1979 -

lots.

Sources of information, 3. Copyright certificateSSSR

Taken into account in the examinationN, cl. C 12 N 1/1, 1971.

Claims (1)

Claim The strain of the fungus Aspergillus nlger L-4 (collection of the Central Museum of Industrial Microorganisms of the Institute VNIIGenetika, collection number TsMPM F-171) “producer of citric acid.