Synthesis of Vitamin B, by Yeast J
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J. M. VAN LANEN, H. P. BROQUIST, MARVIN J. JOHNSON, I. L. BALDWIX, AND W. H. PETERSON University of Wisconsin, Madison, Wis. When living yeast cells, growing or nonproliferating, are aerated in the presence of thiamin, most of the thiamin is taken up by the yeast cells. If the amount of thiamin present is in excess of the quantity necessary to raise the thiamin content of the yeast to about 3 mg. per gram, a large part of the thiamin is not absorbed. It is possible, however, to obtain yeast containing 6 mg. thiamin per gram. When yeast is grown in the presence of the pyrimidine and thiazole portions of the thiamin molecule, thiamin is synthesized by the yeast. The efficiency of the conversion is 70 to 90 per cent, provided the thiamin content of the yeast does not
HE stpimulatoryeffect of thiamin on yeast growth was demonstrated by Williams and Roehm (11) in 1930.
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That the pyrimidine and thiazole constituents of thiamin could serve as well as thiamin for yeast growth was shown by Schultz, Atkin, and Frey (8). With either one or both constituents, similar observations with fungi (6, 7) , bacteria (S),and experimental animals (1, 6) have been reported. Pavcek, Peterson, and Elvehjem (4) found that bakers’ yeast absorbed thiamin from the medium and resynthesized thiamin destroyed by autoclaving. They were able to increase the thiamin content of bakers’ yeast by adding thiamin to the medium. Their investigations also demonstrated that a low temperature of incubation and a grain and molasses medium favored thiamin synthesis, while synthetic media, and small inocula were unfavorable to thiamin synthesis. The effect of factors influencing the synthesis of thiamin from pyrimidine and thiazole by bakers’ yeast has not been reported. Since this investigation mas completed, a patent (9) has appeared describing the produrtion of yeast of increased vitamin B, content by the addition of pyrimidine or pyrimidine and thiazole to the medium. High-thiamin bakers’ yeast, containing 660 micrograms thiamin per gram (dry basis), has been on the market for some time.
Methods Cultures employed were as follows: isolations from three commercial high-thiamin bakers’ yeasts, various strains obtained from the Wisconsin culture collection, and commercial low-thiamin bakers’ yeast obtained on the market as needed. One strain from commercial high-thiamin bakers’ yeast was used in most of the experiments and is here called “stock yeast”. The medium contained the following: beet molasses 36 cc., steep water 7.5 cc., diastatic malt extract (obtained from Red Star Yeast and Products Company) 21.0 grams, diammonium phosphate 1.0 gram, and distilled mater to make 1 liter. They could not be sterilized together without excessive precipitation and hence were treated as follows:
rise above 800 micrograms per gram. In yeast grown without aeration, the efficiency of conversion is low. Nonproliferating yeast cells, under aeration, will convert pyrimidine and thiazole into thiamin almost quantitatively. The presence of spent wort is beneficial. The pyrimidine portion of thiamin is more readily synthesized by yeast than the thiazole portion. None of a large number of compounds tested was able to replace either pyrimidine or thiazole in thiamin synthesis by yeast. Of a number of yeast species and strains, bakers’ yeast showed the best ability to synthesize thiamin from the intermediates.
Except for the phosphate, the ingredients in double the final concentration were adjusted to p1-I 5.0 potentiometrically and steamed for 30 minutes. After cooling, the medium was clarified by Buchner filtration through a ‘/{-inch layer of diatomaceous filter aid. After dilution the medium was again steamed for 30 minutes in 200-cc. quantities in 500-cc. Erlenmeyer flasks. The flasks were previously autoclaved for 30 minutes at 120’ C. The solution of phosphate, adjusted to p H 5.0 with hydrochloric acid and autoclaved for 15 minutes at 120” C., was added aseptically at the time of inoculation. This medium was used in all experimmts except those involving the use of a synthetic medium. The pyrimidine (2-methyl - 5 - ethosymethyl-6- aminopyrirnitline) and thiazole (4-methyl-5,~-hydroxyethylthiazole) (obtained through the courtesy of Merck and Company), made up in solutions containing either 15 or 20 millimoles per liter, were steamed for 15 minutes and added asepticrilly nt the time of inoculntion. Other materials studied were either filtered through a Berkefeld filter or autoclaved and added at the time of inoculation. The medium contained about 3.9 per cent total sugar and 0.16 per cent nit ogen. The inoculum for 200 cc. of medium was 10 cc. (about 0.3 gram of moist cells) of a 24-hour culture. Unless otherwise statetl, all cultures were shaken continuously in a mechanical shaker. After 24-hour incubation a t 30” C. with continuous shaking, the medium yielded about 30 grams of moist cells per liter. Moist cells contained about 25 per cent dry matter. I n all experiments duplicate flasks were employed. The yields reported are on the basis of dry weight of cells per liter of medium. The conversion of reducing sugar to dry yeast was about 20 per cent. This conversion could be increased by using more vigorous methods of aeration or by lengthening the period of incubation. Thiamin mas determined by a modification of the method of Emmett, Peacock, and Brown ( 2 ) . I n the modified method, centrifugation was substituted for filtration, and the color was read in a photoelectric colorimeter. The values in the tables are averages of duplicate determinations. The maximum difference between duplicate determinations was 10 per cent, the average difference less than 5 per cent. 1244
October, 1942
INDUSTRIAL AND ENGINEERING CHEMISTRY
Absorption of Crystalline Thiamin by Living Yeast 4
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TABLE 111. EFFECTOF VARYING CONCENTRATIONS OF PYRIMIDINE AND THIAZOLE ON SYNTHESIS OF THIAMIN BY YEAST
If synthetic thiamin is added to a suspension of growing or resting yeast cells, it, is absorbed by the cells in large amounts. The data in Table I show that growing cells or resting cells suspended in spent medium absorbed most of the added thiamin until the thiamin content of the yeast reached 2000 micrograms per gram. By addition of very large amounts of thiamin to the medium, yeast containing 0.G per cent thiamin could be produced. Since all yeast samples were washed three times by centrifugation from saline before assay, only thiamin actually within the cell was measured.
Yield of Dry Yeast
Thiamin Content of Yeast, uG./G. .~ 55 2Gl
Conversion of Intermediates,
Pyrimidine Thiazole Millimole/
