HU185652B - Process for producing riboflavine with microbiological method - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a microbiological process for the production of riboflavin by culturing a microorganism capable of producing riboflavin from the Bacillus stubtilis species in a medium of submergence in air in a medium containing carbon and nitrogen sources, mineral salts and growth promoters. The method according to the invention is carried out by using a strain of WNIIGenetics-304, CMJM B-2116 or deposited under B-2117 of WNIIGenetics 304a, CMJM B-2116 as a microorganism capable of producing Bacillus stable species. -1-

Description

The present invention relates to a process for the production of riboflavin by microbiological processes.

Riboflavin (vitamin Bj) is widely used in agriculture, the pharmaceutical and food industries. In agriculture it is mainly used as a feed additive for animals.

A process for the production of riboflavin by cultivation of Eremothecium ashbyi fungi under submerged conditions is known. According to Soviet certificates 389132 and 368 302, E. ashbyi fungi produce riboflavin up to 2000 mg / l in cultured in soy medium containing hydrol (starch waste, glucose content about 50%) and corn jam for 96 hours.

It is known to increase the productivity of fungal strains in media rich in vegetable oils, fatty acids, glycerol or lecithin. According to German Patent No. 1767,260, E. ashbyi cultures cultured in medium containing more than 3% glucose, linoleic acid, butyric acid or glycerol, ammonium glutamate, vitamins produce 3800 mg / L of riboflavin. According to German Patent No. 2,028,355, E. ashbyi fungi are cultured in a medium containing 1-5% glucose, vegetable oil, 5% collagen, 0.5-5% lecithin, corn jam. Under these conditions, 4500-5000 mg / L riboflavin is produced.

The major advantage of producing microbiologically riboflavin with E. ashbyi culture is its high biosynthetic activity. In contrast, fungal strains capable of producing riboflavin have undesirable properties. These include the long growth period, which increases fermentation time, and the sensitivity to foreign microflora contamination and media composition constraints that require many expensive and inaccessible components. Antibiotics are added to the fermentation medium to prevent foreign microflora from being formed in the culture of E. ashbyi, but these significantly increase the cost of fermentation. In the cultivation of E. ashbyi, the components used in the fermentation medium, such as various fats, glycerol, fatty acids, lecithin, are difficult to access and expensive.

According to another known process (U.S. Patent No. 3,900,368), riboflavin is produced by culturing Bacillus subtilis species under submerged conditions. In this method, induced mutants of B. subtilis FERM-P 1657 and FERM-P 2292 are used, especially mutants resistant to high levels of purine and purine analogs or purine auxotrophic mutants. These mutants are cultured in media containing 8% glucose, mineral acid, growth promoter component, yeast extract, peptone, glutamic acid, RNA (ribonucleic acid) preparation, purine or purine analogs. During the 44 hour fermentation, 560 mg / L riboflavin is produced in the culture fluid. The advantage of this process is the rapid growth rate of the bacteria and hence the reduction of the fermentation time. However, the activity of the Bacillus subtilis mutant strains used in the method is low. The process also has the disadvantage that the components used in the medium, such as RNA preparations, purine, purine analogs are difficult to access and consequently difficult to apply on a commercial scale. It is an object of the present invention to provide a microbiological process for the production of riboflavin which can shorten the fer15 mentation time and thereby reduce its cost without reducing the yield of the product.

It is an object of the present invention to provide a novel strain having high flavinogenic activity for the production of riboflavin of the species Bacillus subtilis.

SUMMARY OF THE INVENTION The present invention relates to a process for the production of riboflavin by submerged cultivation of microorganisms capable of producing riboflavin of the B. subtilis species in the presence of air in a medium containing carbon and nitrogen sources and the required mineral salts. The riboflavin producing microorganisms of the present invention are strains of B. subtilis deposited under number WNIIGenetics-304, CMJM P-2116 or deposited under the numbers WNIIGenetics-304a, CMJM B-2117.

These strains were deposited on July 22, 1980 at the Central Museum of Industrial Microorganisms of the WNIIGenetics Institute. The designation "WNIIGenetika" in the name of the tribes is "Industrial Mík. Nationwide Research Institute on Genetics and Selection of Organisms.

"CMJM" stands for Central Museum of Industrial Microorganisms.

The known strains of B. subtilis SHWG serve as starting cultures of the said production strains, from which independent mutants are obtained by which riboflavin can be synthesized: ros ^R - resistance to roseflavin, rib ^c - mutation in the regulatory gene, resistance to AGR - 8-azaguanine . Subsequently, the ros ^R -rib ^c -AGR triple mutant was prepared by genetic engineering, which was then treated with nitrosoguanidine, nitrosomethylurea, irradiated with ultraviolet radiation, and the active variants were selected. This gives the two strains.

WNIIGenetics - strain 304 is a prototroph, WNIIGenetics - strain 304a is an auxotrophic microorganism, their growth needs have not been determined. Both strains are oligo-spore-forming. The number of spores is 3 to 4 orders of magnitude lower than the initial 5 cultures. The cultivation-morphological properties of the WNIIGenetics-304 and WNIIGenetics-304a strains capable of producing riboflavin are shown in the table.

185,652

Spreadsheet

specific

WNlIGenetics — 304 strains

WNlIGenetics — Strain 304a

Morpho-2-3 pm long spore-forming gram-positive bacilli logy under the microscope

Morphology in different media

Meat peptone agar (FPA)

Meat peptone bouillon (FPB)

Pelleted culture on minimal medium with glucose

At 28-37 ° C, it forms star-like cultures 3 to 4 mm in diameter on 1-2 days with non-smooth edges, smooth surface, shiny, yellowish odor with moderate growth, mainly on medium without shaking on liquid surface in the form of film on day 2 of growth 2-3 mm in diameter on minimal medium no cultures, light yellow in color, fluoresces yellow under ultraviolet rays, cultures round, wide smooth, shiny

Physiological properties Increase at 20-42 ° C (optimum 37 ° C), pH 5.0-9.0 (optimum 7.0-8.0) carbon sources: good use of glucose, sucrose, starch alcohols: ethanol and sorbitol are utilized by nitrogen sources: well assimilates ammonium salts, urea, nitrate liquefies gelatin

The process of the invention is generally carried out as follows.

The WNlIGenetika-304 and WNlIGenetika-304a strains were prepared for 1 to 2 days in 30 agarized Hottinger's medium (Birger Μ. FPA) was cultured at 28-37 ° C. The cell suspension is then transferred to a medium containing 35 molasses, dry biomass of feed yeast and mineral salts, or in flasks containing purified potato chips or meat-peptone bouillon. The inoculum was cultured on a 200 rpm shaker for 17-24 hours. The vaccine

5 to 10% are then added to the main fermentation medium. The fermentation was carried out in an Erlenmeyer flask on a shaking table for 46-50 hours at 37 ° C and pH 6.5-7.5 (at the start of the fermentation). The medium used includes carbon sources such as glucose, sucrose, raw sugar, molasses, starch, mineral nitrogen sources such as ammonium salts, urea, nitrate, phosphorus salts such as potassium phosphate, ammonium phosphate and other salts of manganese, dichlorod, manganese sulfate, and growth promoters such as feed yeast biomass, feed yeast hydrolyzate and autolysate, edible yeast, yeast extract 55, corn and soybean meal, corn stearate, casein hydrolyzate. After 46-50 hours of fermentation, the culture fluid contains 700-1000 mg / L riboflavin.

Riboflavin preparation according to the present invention uses 1000mg / l of riboflavin in culture medium, producing 40% more activity than θ, in culture flasks over 46 to 50 hours using the indicated strains in synthetic or complex nutrient media.

The activity of a B. subtilis culture used according to U.S. Patent No. 900,368. In addition, the strains of the invention may be well grown in simple medium containing inexpensive components.

Comparing the process according to the invention with the process using E. ashbyi fungus as a producer of riboflavin, it can be stated that due to the acceleration of fermentation, the process according to the invention is 1.5 to 2 times more economical as bacterial cultures grow faster than mushroom cultures. It should not be underestimated that the B. subtilis culture is less sensitive to foreign microflora compared to fungi, which is particularly important because it does not require the addition of an antibiotic to the fermentation medium and makes the industrial process much cheaper.

The invention is illustrated by the following examples.

Example 1

Cultured on inclined meat peptone agar (FPA) for 24 hours

Cultures of B. subtilis WNI1Genetics-304 were inoculated into a liquid seed medium containing 6% molasses, 1% fodder yeast dry biomass and the following mineral salts. (The inoculum medium is obtained by diluting the main fermentation medium twice with sterile water. Cultured at 37 ° C. The resulting vaccine is added in an amount of 5% to the main fermentation medium of the following composition:

-3185652 Molasses 12% by weight of dry yeast forage yeast 2% by weight of ammonium sulphate [(NHJj SO ₄ ] 0,2% by weight of potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0,6% by weight of potassium hydrogen phosphate (K ₂ HPO ₄ ) 1.4% by weight magnesium sulfate (MgSO ₄ ) 0.01% by weight sodium citrate (Na ₃ C ₆ H ₉ O ₇ ) 0.1% by weight pH - 7.0-7.2

The fermentation was performed on a shaking table at 200 rpm in a 750 ml Erlenmeyer flask (25 ml medium) at 37 ° C. The fermentation was carried out under the specified conditions for 48 hours, so that the culture fluid contained 800 mg / L riboflavin.

Example 5

The culture, seed medium, fermentation conditions are as in Examples 1, 2, 3 or 4. The difference is in the composition of the main fermentation medium, which is as follows:

sucrose (or crude sugar) 10% by weight of dry yeast forage yeast 3% by weight of ammonium sulphate [(NH ₄ ) ₂ SO ₄ ] 0,2% potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0,6% potassium hydrogen Phosphate (K _: HPO ₄ ) 1.4% Magnesium Sulfate (MgSO ₄ ) After 0.01% w / v fermentation, the culture medium contains 700 mg / l riboflavin.

Example 2

The fermentation was carried out as described in Example 1 except that the strain WNIIGenetics-304a was used. After 48 hours of fermentation, the culture medium contains 1000 mg / L riboflavin.

Example 3 The main fermentation medium and fermentation conditions are as in Example 1, using WNIIGenetics-304 as the producer. Compared to Example 1, the difference is in the composition of the seed medium. The inoculum medium is prepared as follows. The peel of the potato is peeled, cut into pieces, poured over a small amount of water and then sterilized at a pressure of 0,8 · 10 ⁵ · 1 · 10 'Pa. The inoculation medium was inoculated from a meat peptone agar (FPA) slant culture and the culture was grown in a 750 ml Erlenmeyer flask (volume of medium on a 50-100 ml shaking table for 20 hours at 37 ° C.) The main fermentation medium was inoculated with 10% inoculum. and fermentation was carried out as described in Example 1. After 48 hours of fermentation, the culture fluid contained 900 mg / L roboflavin.

Example 4

The culture was carried out in the same manner as in Example 3 except that B. subtilis strain 55 WNIIGenetics-304a was used. After 48 hours of fermentation, the culture medium contains 900 mg / L riboflavin.

weight% 0.5 weight% 0.02 weight% 0.2 weight% 0.6 weight% 1.4 weight% 0.01 weight% 0.1 weight%

Example 6

A 24-hour culture of WNIIGenetics-304 strain cultured on oblique meat-peptone agar was inoculated with meat-peptone bouillon inoculum and the inoculum was prepared at 37 ° C for 10 hours on a shaking table. The inoculum is added in 10% main fermentation medium with the following composition: glucose yeast extract casein or peptone hydrolyzate ammonium sulfate [(NH ₄ ) _; SO ₄ ) potassium dihydrogen phosphate (KH ₂ PO ₄ ) potassium hydrogen phosphate (K _; HPO ₄ ) magnesium sulfate (MgSO ₄ ) sodium citrate (Na ₃ C ₆ H ₉ O ₇ ) pH-7.1 -7.2

The fermentation was carried out on a shaker at 200 rpm for 48-50 hours at 37 ° C. After 48 hours of fermentation, the culture medium contains 1000 mg / L riboflavin.

Claims (2)

Patent claims 1. A microbiological process for the production of riboflavin by submerged cultivation of a microorganism of the Bacillus subtilis species capable of producing riboflavin in the presence of air in a medium containing carbon and nitrogen sources, the required mineral salts and growth promoters, characterized in that Bacillus subtilis - strain 304, CMJM B-2116 or strain WNIIGenetika-304a, CMJM B-2117 are used. The method of claim 1, wherein the carbon source is cultured in a medium containing sucrose, glucose or molasses, a growth biomass or yeast extract as a feed yeast or edible yeast.