DE1091286B - Process for obtaining vitamin B. - Google Patents

Description

Method for obtaining vitamin Bi. The invention relates to a Process for the production of VitatninBl, by cultivating thermophilic organisms suitable culture media.

So far, VitaminBl. Obtained through the action of bacteria and fungi using a variety of strains, nutrient media and fermentation conditions. The organisms used for this purpose were each of the types referred to as psychrophilic or mesophilic, which show optimal growth rates at temperatures between human body heat, ie about 37 ° C, down to 15 to 20 ° C. However, only low yields were achieved using these organisms. Since other undesirable organisms also grow well within the temperature range mentioned, both sterilized culture media had to be used in all cases and extensive precautions had to be taken to maintain the sterility of the media during fermentation.

The inventive method for the production of Vitamin Bl, by incubating a manufacturing with a vitamin B12 microorganism inoculated nutrient medium is now characterized in that a thernlophiler, Vitamin B ". Producing bacillus, in particular a strain of Baeillus stearotherrnophilus, Bacillus coagulans or Bacillus circulans used .

The use of thermophilic bacteria, i.e. H. of bacteria, which are best grown between about 45 to 75 "C , for the production of vitamin B, 21 has the great advantage that other types of organisms which do not lead to the production of vitamin B12 or are undesirable for other reasons, within the With the method according to the invention it is therefore no longer necessary to sterilize the culture media used beforehand or to keep them sterile during the growth period and to avoid any contamination in the known processes.

Another advantage of the inventive method is based on that the time required to produce a certain amount of vitamin B, 2 is essential is shorter than with the previously used methods.

The application of thermophilic bacteria in accordance with the The method according to the invention also enables the use of specially developed methods Culture media adapted to the needs of these organisms. The selective one Adding nutrients and eliminating certain components that are necessary for this Types of organisms are not required, led to the development of new culture media, which are less expensive and easier to manufacture.

According to the invention, a nutrient medium, preferably the above-mentioned new nutrient medium, is inoculated with a vitamin B 2-forming strain of thermophilic organisms and growth is carried out at a temperature between about 45 and 75 ° C. under aerobic immersion conditions, preferably with stirring, until a maximum content of Vitamin B, 2 is obtained, whereupon from the medium by means of known methods, such as. B. by heating, adding acid or evaporation, a concentrate of vitamin Bl. Is obtained, which can be used as an additive to animal feed. Other known cleaning methods can also be used, e.g. B. Contacting with absorbents.

The thermophilic organisms used according to the invention for the production of vitamin B 12 are strains of various thermophilic bacilli. In a preferred embodiment, the species Stearothermophilus and Coagulans are also included. The species mentioned fall under the current classification system of the Bergeys Manual, and various specific strains have been isolated which produce vitamin B 12, but with different degrees of effectiveness. According to the invention, other types such. B. Circulans, Subtilis, Macerans and Sphaericus can be used. Certain strains of these species are known to exhibit at temperatures above 45 'C good growth and are as thermophiles' denotes (MB Allen, 1953, The thermophilicity aerobic sporeforming baeteria Bacterial. Ev., 17, pp 125-173, and N. R Smith, R. E. Gordon and FB Clark, 1952, Aerobic sporeforming bacteria. US Dept. Agr. Monograph No. 16). It has been found that strains isolated from hot springs, geysers, and the like in the United States and Cuba have particularly good properties for producing vitamin B ".

It is noted that among the bacilli mentioned above, Coagulans and Stearothermophilus are real thermophiles which grow at higher temperatures than the other mentioned species, which have often been regarded as adapted strains of the mesophilic bacteria which are still actively growing at temperatures between 45 and 55 ° C demonstrate. The species Coagulans and Stearothermophilus, which still grow well at 60 or 75 ° C., have certain advantages over the other species mentioned and are preferred for the process according to the invention. Of the species that have adapted to growth at higher temperatures, the Circulans group and in particular a specific strain, which will be described in more detail below, is particularly suitable because at relatively high temperatures, i. H. at about 55 ° C, high yields of vitamin B12 can be achieved.

The thermophilic bacteria, which were obtained from the above-mentioned thermal sources and show extremely good properties for the production of vitamin Bl., Are z. B.: Bacillus stearothermophilus 194, deposited in the "American Type culture collection" and designated as ATCC 12986; Source: Yellowstone Geyser Mud; Bacillus coagulans 406, deposited in the American Type Culture Collection and designated as ATCC 12989; Source: Yellowstone Geyser Mud; and Bacillus coagulans 416, deposited in the American Type culture collection and referred to as ATCC 12990; Source: Old Faithful Geyser Water. In addition, it was found that thermophilic bacterial strains isolated from animal excrement also had good properties for the production of vitamin Bi. A particularly good example of this class of organisms is: Bacillus circulans CI 372, deposited in the "American Type culture collection" and designated as ATCC 12994; Source: cow dung. By means of the four strains mentioned above, yields of vitamin B of 3 to 6 mg per liter can be obtained in non-synthetic media and from 1 to 2 mg per liter in synthetic media.

In addition to the strains already mentioned, numerous other strains of thermophilic organisms have also been isolated and investigated which, although less effective, still lead to the production of vitamin Bi. In yields of at least 0.5 mg per liter of the medium. These strains are: Bacillus stearothermophilus 1, 27, 32, 35, 63, 67, 72, 79, 90, 109, 137, 148, 149, 150, 152, 155 (ATCC 12987), 160 (ATCC 12988), 167, 189, 195, 197, 202 and 203; Bacillus coagulans Co 413, Co 432, Co 439, Co 467, Co 491; Bacillus subtilis ST-T-381 (ATCC 12993); Bacillus sphaericus SP-312 (ATCC 12992) and Bacillus macerans M-314 (ATCC 12991). The strains preferred for the production of Vitamin Bi. Are described on the basis of their morphological, cultural and biochemical properties as follows: Bacillus stearothermophilus 194 If this Bacillus is grown at a temperature between 45 and 70 ° C , it has the following properties: Vegetative rods, 0.5 to 1.0 11 to 2 to 4 [t, mobile (motile), gram-positive, older rods stain gram variably; Swollen sporangia; Spores 1 to 2 [t to 1 to 2 li, sometimes ovoid, spore membrane usually stainable; Colonies indistinguishable when growing on agar. Nutrient agar slants, growth is opaque and spreading; Nutrient broth evenly cloudy; Utilization of citrate negative; Hydrolysis of starch positive -, production of acetylinethylcarbinol negative; pu value of the broth 5 to 7, optimally between 6 and 6.5; Hydrolysis of gelatin positive; Fermentation tests: glucose, acid without gas in the presence of amino acid. Temperatures for growth: good growth at 45 to 70 ° C, optimal growth at around 55 ° C in a chemically defined medium or in nutrient broth semi-diluted with water, supplemented with 0.5 % casein hydrolyzate. Storage conditions: Agar slants made from 0.511 / o polypeptone (Baltimore Biological Laboratories) + 0.5% meat extract + 2.5'Dlo agar can be stored at 4 ° C. for more than 1 year without deterioration in the growth of the above-mentioned organism in the specified Temperature range occurs. Baeillus circulans CI 372 Properties: vegetative rods, 0.3 to 0.7 11 to 1.5 to 5 [t, rounded ends, mobile (motile), gram-negative, sometimes gram variable; Swollen sporangia; Spores kidney-shaped; Colonies spreading opaque; Nutrient agar slants, growth spreading and dense; Nutrient broth slightly cloudy; Utilization of citrate negative; Hydrolysis of starch positive; Acetylmethylcarbinol production negative; Fermentation tests: acid without gas from glucose, arabinose, xylose and sucrose; Hydrolysis of gelatin positive; -production of indole negative; Temperatures - for limited growth from 28 to 451 C, for good growth from 45 to 60 'C. Bacillus coagulans 406 and 416 Properties: Vegetative rods, 0.5 to 1.5 [t to 2 to 5 li, mobile (motile) , gram positive; Sporangia swollen at No. 406, not swollen at No. 416; Colonies opaque; Nutrient agar slants irregular, opaque, flat; Utilization of citrate negative; Hydrolysis of starch negative at 55 ° C, positive after 2 weeks of incubation; Fermentation tests: acid from glucose without gas from glycerine and sucrose; Hydrolysis of gelatin weak; Temperatures, optimal growth of Borth strains at 45 to 65 ° C, some growth at 37 to 45 ° C; Storage conditions: on agar slants made of 0.5 % polypeptone + 0.51% meat extract + 2.5% agar, the organisms can be stored for more than 1 year at 4 ° C. without deterioration in growth in the specified temperature range. In a non-synthetic culture medium, strain 406 produces 2 to 3 mg vitamin B12 per liter, strain 416 3 to 6 mg per liter.

The various strains mentioned above are very similar to one another in their general morphological and biochemical properties. They are highly aerobic and show very little growth under anaerobic conditions. The optimal temperature range for their growth is between about 45 and 65 ° C, whereby the Bacillus stearothermophilus 194 still shows good growth at 75 ° C. The incubation time is between 8 and 72 hours.

It has been found that a pH of 5 to 8.5 is suitable for obtaining vitamin Bi. to achieve the best result, however, a pH value of about 6 to 7.5 is preferred, which can rise to about 8 to 8.5 towards the end of the incubation time. If the p11 value is not kept at this level during active growth, the yield of vitamin Bi. Is reduced. While the pH can be maintained by the addition of any suitable buffer, it has been found that a buffer made from a combination of aconite and triethanolamine works best for this purpose because it is not metabolized by the organisms. For this reason, no phosphate buffer is expediently used, since this is absorbed by the metabolism, possibly with loss of the buffering effect.

The sterility which is generally required for the growth of vitamin B12-producing organisms is not required for the growth of the thermophilic organisms which can be used according to the invention, although sterile conditions can of course also be maintained if necessary. The effectiveness of the thermophilic growth conditions in avoiding contamination by mold and other foreign organisms can be proven by experiment. These were carried out by exposing an agar medium, which had been sown with cultures of these thermophiles, to the air of the laboratory at room temperature for 1 day. After 1 day, the plates were incubated at 37, 55 and 75 ° C. After 18 hours of incubation, the plate, kept at a temperature of 37 ° C., showed strong growth of mold and other contaminants. However , the plates kept at 55 and 751 C showed only the growth of thermophiles and no impurities.

As already mentioned, the thermophilic, vitamin B1-producing organisms can be grown either on non-synthetic or on synthetic culture media by keeping a supply of media ready and working under immersion conditions, with the ventilation necessary for aerobic conditions being maintained. In any case, it is necessary that the medium used contain all the components required for growth, i. H. Mineral salts, trace elements, amino acids and the like. These components are partially supplied by the already known nutrient media, preferably by media such as cereal softening liquid, barley, potato, rice and other cereal concentrates. Riboflavin .................... 5 ylg Never ....................... 819 ylg Pantothenic acid ............... 23.8 Ilg Pyridoxine .................... 19.1 ylg Biotin ....................... 0.125 1 / g When preparing a non-synthetic culture medium, it is preferred to use grain softening fluid. This generally has the following vitamin content: It also has the following amino acid content: % Amino acid Amino acid (hydrolyzed) Leucine ..... .................... 4.78 Isolenein ....................... 2.77 Valine .................. * ....... 2.81 Glutamic acid ................. 7.28 Threonine 2.54 Lysine .... 1.80 Arginine 2.23 Histidine 2.18 Proline 3.39 Phenylalanine ................... 2.10 Methionine ..................... 0.00 Aspartic acid ................ 1.39 Cystine ......................... 0.91 Alanine ... 15.33 Tyrosine 0.78 Ammonia ............ ......... 1.34 A non-synthetic medium suitable for the growth of thermophilic bacteria can be made from the following components: Grain softening liquid ........ 5 cc Sodium cyanide ................ 2 mg Cobalt ion (like Co S 04 - 7 H2 0) 4 mg Citric acid .................. 100 mg Triethanolamine ................ 300rng Potassium hydroxide to ........... p11 value 6.0 Distilled water to ........ 100 ccm Although sterilization is not required, the medium described above can be sterilized by autoclaving it for 30 minutes at a temperature of about 118 to 120 ° C. under a pressure of 1.05 kg / cm 2.

The following examples serve to illustrate the process according to the invention: Example 1 A culture medium hergestell t by was added 800cc of the broth described above in four Roux bottles, each of which had a capacity of 4 1, and then the broth with Bacillus coagulans 416 (ATCC 12990) . The growth was carried out under aerobic conditions. When the bottles were positioned so that they had a maximum air surface area, no stirring was required. After 18 hours at 55 ° C., the p11 value of the broth was fixed at 4.5 with citric acid. After the incubation period had ended and the pH had been regulated, 1.0 g of sodium metabisulphite was added per cubic centimeter of broth in order to ensure reducing conditions to protect the protein-bound vitamin B 12 released. The protein-bound vitamin B "was then released by treating the broth together with the bacilli in the autoclave for 30 minutes at 118 ° C. The vitamin B" was made by modifying the proposed USP method (US Pharmacopeia, XIV, 3 . Supplement, pp. 15 to 19) , three different "%" test organisms being used for comparison purposes. These organisms were: Lactohaeillus leichmannii ATCC 4792, Euglenia gracilis (Z-Starnm) and Ochromonas malharnensis (Pringsheim strain). The average The yield was between 3 and 6 mg vitamin B " per liter of the culture medium. The vitamin Bi. Was obtained from the broth using conventional methods. The autoclave-treated material was then drum- dried at a temperature of 40 ° C. and made a supplementary feed for animals. It is noted that during the growth period the yield approached the maximum after 8 hours. At this point in time the broth still has the initial pH value of 6. After 12 hours the amount B.2 produced is slightly higher than after the end of 8 hours, and the pI value is 7.5. The highest yield is reached after about 18 hours when the pH is 8 to 8.2 . Although growth can continue for up to 72 hours, the pi value should never exceed 8.2 for maximum yields. Example 2 A medium was prepared by adding 800 cc of the broth described above to four 41-capacity Roux bottles and inoculating with Baeillus stearothermophilus 194 (ATCC 12986). The growth was carried out under aerobic conditions. If the bottles were set up so that they had as large an air surface as possible, no stirring was necessary. After 18 hours at 65 ° C., the pH of the broth was fixed at 4.5 with citric acid. After the incubation time had ended and the pH had been regulated, 1.0 mg of sodium metabisulphite per ccm of broth were added in order to ensure reducing conditions to protect the protein-bound vitamin Bi. The protein-bound vitamin B 12 was then released by autoclaving the broth together with the bacilli for 30 minutes at 118 ° C. The vitamin B12 content was determined by modifying the proposed USP method (US Pharmacopeia, XIV, 3rd amendment, pp. 15 to 19) , three different test organisms, namely Lactobaeillus leichmannii ATCC 4792, Euglenia graeilis (Z- Starnm) and Ochromonas malhamensis (Pringsheim-Stamin) were used. The average yield was between 2 and 3 mg of vitamin B1 per liter of the culture medium. The vitamin B 12 was obtained from the broth using known methods. Example 3 A medium was prepared by mixing 800cc of the broth in the above-described four Roux bottles -and optionally having a capacity of 4 1 were inoculated with Bacillus coagulans 406 (ATCC 12989). The growth was carried out under aerobic conditions. If the flanges were set up in such a way that they had as large an air surface as possible, no stirring was necessary. After 18 hours at 55 ° C. , the pH of the broth was fixed at 4.5 with citric acid. After the incubation time had been reduced and the pH had been regulated, 1.0 mg sodium metabistilite was added per cubic centimeter of broth in order to ensure reducing conditions to protect the released protein-bound vitamin Bi. The protein-bound vitamin B 12 was then released by autoclaving the broth together with the bacilli for 30 minutes at 118 ° C. The vitamin content was determined by modifying the proposed USP method (U. S. Pharrnacopeia, XIV, 3rd amendment, pp. 15 to 19) , using three different test organisms for comparison purposes, namely Lactobacillus leichmannii ATCC 4792, Euglenia gracils (Z strain) and Ochromonas malhamensis (Pringsheim strain). The average yield was between 2 and 3 mg of vitamin B " per liter of the culture medium. The vitamin B 1 was obtained from the broth using known processes.

Example 4 A medium was prepared by adding the broth described in four Roux bottles given 800ccrn with a capacity of 4 1 and were inoculated with Baeillus circulans CI 372 (ATCC 12994). The growth was carried out under aerobic conditions. If the bottles were set up so that they had as large an air surface as possible, no stirring was necessary. After 18 hours at 55 ° C. , the pH of the broth was fixed at 4.5 with citric acid. After the end of the incubation time and after regulating the pI value, 1.0 mg sodium metabisulphite per cubic centimeter of broth was added in order to ensure reducing conditions to protect the protein-bound vitamin B, 2 released. The protein-bound vitamin BJ., Was then released by autoclaving the broth together with the bacilli for 30 minutes at 118 ° C. The vitamin B12 content was determined by -. \ Iodification of the proposed USP method (US Pharmacopeia, XIV, 3rd amendment, pp. 15 to 19) , three different test organisms being used for comparison purposes, namely Lactobaeillus leichannii ATCC 4792 -, Euglenia graeilis (Z strain) and Ochromonas malharnensis (Pringsheim strain). The average yield was between 3 and 6 mg vitamin B " per liter of the culture medium. The 1 \ titarnin Bi. Was obtained from the broth using known methods.

Using yeast extract or L-glutamic acid as a source for the amino acid, a synthetic culture medium was developed according to the invention which is suitable for the production of vitamin B 12 from thermophilic organisms. The trace elements were provided by adding appropriate amounts of a metal mixture of the following composition: Ethylenediaminetetraacetic acid ... 2.5 g Zn S 04 7 H2 0 ................ 17.6 g Mn S 04 H2 (:) ................. 9.22 g CUS04 - 5 H20 ................. 0.98 g FeS 04 (N H4) 2 S 04 - 6 H2 0 .... 0.7 g H3B03 ........................ 01571 g Cos04.7 H20 ................ 0.19 Distilled water to ........ 1000 ccm The synthetic culture medium suitable for use with the organisms described above has the following composition: Ethylenediaminetetraacetic acid .... 0.05 g KH2P04 ....................... 0.02 g Mg S 04.7 H2 0 ................ 0.05 g Mo (like ammonium molybdate) ... 1.0 mg Solution of the mixed metal salts 1.5 cc NH4C1 ......................... 0.03 g Ca-Ion (like cac12) .............. 1.0 mg Aconitic acid (trans) .............. 0.5 g Triethanolamine .................. 1.0 g Sodium acetate 3 H20 ............ 0.04 g Glycerin ........................ 1.0 g Potassium gluconate ................. 0.5 g NaCN ......................... 0.2 mg Sodium metabisulphite ............. 0.1 g Succinic acid .................. 0.05 g Distilled water ............. 100 cc L-glutamic acid can be added to the above mixture in amounts between 0.01 and 1.0 g per 100 ecin culture medium. However, about 0.5 g of yeast extract can also be added per 100 ccm of medium. Additional amounts of sodium cyanide and cobalt ions can also be used. Example 5 800 ng of a synthetic medium to which 1.0 g of glutamic acid had been added per 100 cc of medium and which was inoculated with Bacillus circulans CI 372 were used. The pff value was initially kept at about 6.5 and increased to about 8 towards the end of the incubation. According to the analysis, a vitamin Bi yield of 1 mg per liter of the medium was obtained.

Claims (2)

PATENT CLAIMS. 1. A method for obtaining vitamin B., by incubating a nutrient medium inoculated with a vitamin B12-producing microorganism, characterized in that a thermophilic, vitamin B12-producing bacillus, in particular a strain of Bacillus stearothermophilus, Bacillus coagulans or Bacillus circulans, is used. 2. The method according to claim 1, characterized in that the incubation is carried out under aerobic conditions. 3. The method according to claim 1 and 2, characterized in that the incubation is carried out at a pE value of 6.0 to 7.5 and at a temperature of about 45 to 75 ° C.