Determination of Thiamine in Bread by
the Thiochrome Method A Comparison of Phosphatase-Containing Enzyme Preparations D. F. CLAUSEN
AND
R. E. BROWN, International Milling Company, Minneapolis, Minn.
A
NDREWS and Nordgren (1) have suggested that a great TABLE I. Assays OF BREADS
deal of the difficulty experienced in assaying bread for thiamine by the thiochrome method may be due to the presence of cocarboxylase formed from the free thiamine b y the action of yeast.
Enzymea Takadiastase Takadiastase Clarase Clarase Clarase Clarase Polidase Polidase Polidase No enzyme (blank determination) Takadiastase Clarase Polidase KO enzyme (blank determination) Takadiastase Clarase Polidase Takadiastase Clarase Polidase Clarase Polidase Takadiastase Chase Polidase Mylase P Yeast extract Takadiastase Clarase Polidase Mylase P Yeast extract
These authors raised the incubation temperature and lengthened the incubation time in order to accom lish com lete hydrolysis of cocarboxylase. Lipton and Ekehjem p8) have shown that thiamine can be coupled with phosphate by yeast to give cocarboxylase, and Livshits (9) found that yeast may be capable of synthesizing thiamine. Conner and Straub (3) and Hennessy, Tarshis, and Perlman (6) have shown that several commercial enzyme preparations will quantitatively hydrolyze aqueous solutions of synthetic cocarboxylase. The latter authors also found aluminum, mercuric, and ferric ions inhibiting, ferrous accelerating, and considerable variation in the enzymes. It is well known that optimum concentrations of accelerators are necessary for many phosphatases if they are to operate at maximum velocity. Besides commercial enzyme preparations, which are usually made from mold growths, other phosphatase preparations have been used to hydrolyze cocarboxylase : Hennessy and Cerecedo (4) used a beef kidney extract, Kirch and Bergeim ( 7 ) used a yeast-glycerol extract, and Melnick and Field ( I O ) used a yeast powder.
In view of these investigations it appeared possible that solutions of pure cocarboxylase might not give the same results with phosphatase preparations as would cocarboxylase in natural products with phosphatase preparations, because the
Thiamine Micrograms/p. 3.11 2.56 3.09 2.52 3.15 3.08 3.10 3.28 3.01 1.62
Commercial 2
1.5 1.5 1.5 1.5
3.51 3.03 4.35 1.41
Commercial 3
1.5 1.5 1.5 1.5 1.5 1.5 16 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
3.96 3.42 5.04 1.32 1.32 1.71 3.12 3.33 1.02 1.35 2.40 2.13 1.14 0.98 0.78 1.20 0.99 0.51
Commercial 4 Commercial 5 Laboratory bread enriched with yeast Laboratory bread not enriched
The Aspergillus JEavus was grown on a 1 per cent peptone4.5 per cent sugar-tap water media, dried by an air blast a t
room temperature, and ground up. It was stored out of direct light at room temperature, as were the commercial enzymes. The yeast extract was kept under refrigeration, where it is stable for months (6). The lanthanum hydroxide did not give encouraging results and was abandoned early in the investigation. The bread samples were broken into small pieces and airdried to about 10 per cent moisture, then ground until 99 per cent passed through a 36 grits gauze, and blended carefully. They were stored a t room temperature out of direct light. Four-gram samples were taken for assay, and the results are given on the “as run” moisture basis. Most of the work was done with a commercial bread (commercial bread 1) made by the sponge process and enriched with a concentrate containing iron, thiamine, and nicotinic acid, as well as a yeast food. Such yeast foods usually contain sodium chloride] calcium sulfate, ammonium chloride, and potassium bromate. Table I summarizes the results of 68 assays on this bread and 21 assays on six other assorted breads. The figures given represent averages of results, all of which agrek within 5 per cent.
Procedure Four commercial enzyme preparations and three preparations made in this laboratory were tested, using the thiochrome reaction as an indicator of the power of the enzyme to split cocarboxylase. A considerable range of results was found, and, as was t o be expected, the results on bread did not coincide with those on aqueous solutions of pure cocarboxylase. Variations were also noticed in the results obtained with different kinds of bread. The assay procedure was that of Conner and Straub (3) with minor modifications. The instrument used for the final readings was a Coleman Model 12 electronic photofluorometer. The enzyme preparations were as follows:
La(0H)s solubion
Commercial 1 (sponge process)
Incubation Time b Hours 16.5 1.5 16.5 1.5 20 18 20 1.5 18 1.5
a Assays of 1-gram samples of enzymes indicated that they were essentially thiamine-free. b Moisture loss was found negligible with long incubation, and no correction was necessary.
work done with pure cocarboxylase does not take into account the effect of numerous activators and inhibitors that might become active when the cocarboxylase in natural products is t o be hydrolyzed. The authors were interested in finding a n enzyme preparation that would efficiently hydrolyze the cocarboxylase in bread in a short time and under the conditions of p H and temperature of digestion of the thiochrome method as it is generally prescribed. This enzyme was preferably to be either a readily available commercial preparation or one that is easily prepared in the control laboratory.
Enzyme Takadiastase Clarase Mylase P Polidase Aspergillus pavus Yeast-glycerol extract
Bread
It is apparent here that the polidase gives the best results, whether the time is 1.5hours or overnight, with mylase P giving second best. For commercial bread 1 mylase P and the yeast preparation did quite as well as polidase, as will be seen in Figure 1. I n all cases takadiastase and clarase gave quantitative results only after incubation periods of many hours‘ Using the sponge process bread (commercial bread 1) time curves were run on all the enzyme preparations. The results
Source Parke, Davis B Co. Takamine Laboratory, Inc. Wallerstein Laboratories Schwarz Laboratories, Inc. Grown in this laboratory prepared in this laboratory after Kirch and Bergeim (7) Used in water solution. Suggested by the work of Bamann and Meisenheimer JB), who found that La(0H)a cleaves sodium glyceryl-8-phosphate
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February 15, 1943
ANALYTICAL EDITION
101
the differences existing between various kinds of enzymes, and perhaps also to the presence of varying amounts of activators and inhibitors in different breads. It also seems reasonable to assume that experiments with aqueous solutions of pure cocarboxylase may not give the same results as with cocarboxylase in bread. It is recommended that, in using any enzyme preparation, the enzyme be allowed to incubate for a t least 18 hours in order t o ascertain the shortest incubation time possible with that particular enzyme and bread.
Summary An enzyme was found that gave complete hydrolysis in 1.5 hours on all breads tested when they were assayed for thiamine content by the thiochrome method. Other enzymes FICTJRE 1. TIMECURVESPRODUCED BY ENZYME PREPARA- gave results that varied considerably with the kind of bread TIONS ou SPOA-GE PROCESS COMMERCIAL BREAD1 used. When used with solutions of pure cocarboxylase, all All preparations were 3% suspensions in the buffer, except the mold, but one of the enzymes gave results inconsistent with those obTemperature, 45'-50' C. A , takadiastase: B , which was 1.1%. clarase; C, mylase P ; D ,polidase tained when they were used on bread. These variations are assumed to be due t o inherent differences between various kinds of enzymes and to the presence of varying amounts of are shown in the series of small graphs in Figure 1. The activators and inhibitors in different breads. curves were continued until further hydrolysis gave no more thiamine, with one exception. It is evident from these graphs that only polidase, mylase P, TABLE11. HYDROLYSIS OF AQCEOUSSOLUTIONS OF COCARand the yeast extract gave complete hydrolysis in 1.5 hours, BOXYLASE and since the yeast extract and mylase P failed to give quanti(Containing 10.0 and 12.5 pg. of cocarboxylase per 50-ml. aliquot) tative results on the laboratory breads (see Table I ) it follows Incubation Enzyme Time Recovery that only polidase can be depended upon t o give good results Hours % in 1.5 hours on all the breads tested. It cannot be predicted Polidase 0.25 100.0 that it would likewise give quantitative results on any bread 2 100.0 Polidase 3 100.0 Polidase tested. The Aspergillus jlavus preparation followed closely 2 2.5 Takadiastase the curves set by takadiastase and clarase. Since it is difficult 2 50.0 Takadiastase 3 10.0 Takadiastase to prepare, no further work was done with it. Clarase 2 100.0 Clarase A concentration curve was run, using polidase and comMylase P mercial bread 1 (Figure 2). It is apparent from this graph that Mylase P Yeast extract it is inadvisable to use concentrations of polidase below 3 per Yeast extract Yeast extract cent in the buffer when the incubation time is 1.5 hours and Blank (no enzyme) the temperature 45" to 50" C.
Acknowledgment
FIGURE 2. CONCENTRATION CURVE WITH POLIDASE Incubation time 1.5 hours, temperature 45' to 50' C.
To compare the action of the various enzyme preparations on aqueous solutions of pure synthetic cocarboxylase, a series of experiments was undertaken using water solutions of cocarboxylase made 0.04 N with sulfuric acid. The buffer solution of enzyme was added as usual. Some 125 assays revealed such variations that no attempt was made to draw any definite conclusions as to the relative merits of the enzymes. Some typical results are shown in Table 11. Only polidase gave quantitative recovery consistently. The causes for this, while unknown are assumed to be inhibitors and accelerators that appear in varying amounts as contaminants in the apparatus. It seems reasonable to conclude from these experiments that variable results with enzymes used t o hydrolyze cocarboxylase in bread-for example, the variation in the results obtained with mylase I' and the yeast extract-may be attributed to
The authors wish to express their gratitude to C. S. French, Department of Botany, University of Pvlinnesota, and E. R. Kirch, College of Pharmacy, University of Illinois, as well as to E. N. Frank of the International Milling Company, Minneapolis: who read the manuscript and gave many valuable suggestions, Thanks are also expressed to F. M. Parker of Merck and Company, who donated the cocarboxylase, to the enzyme manufacturers who donated samples of their products, to Charles Drake of the Department of Bacteriology, School of Medicine, University of Minnesota, who provided the culture of Aspergillusjlavus, and to Leslie G. Wicklund of the International Milling Company, who ran moisture and other determinations.
Literature Cited (1) Andrews, J. S., a n d Nordgren, R o b e r t , Cereal Chem., 18, 686 (1941). (2) B a m a n n , E., a n d Meisenheimer, M., Be?., 71B,1711 (1938). (3) Conner, R. T., a n d S t r a u b , G. J., IND.ENG.CHEM.,ASAL. ED., 13,380 (1941). (4) Hennessy, D. J., a n d Cerecedo, L. R., J . Am. Chem. Soc., 61,179 (1939). (5) Hennessy, D. J., Tarshis, R., a n d P e r l m a n , C., Vitamin program, l O l s t Meeting of t h e A . C . S., -4tlantic C i t y , N. J., 1941. (6) Kirch, E. R., private communication, 1942. (7) Kirch, E. R., a n d Bergeim, O., J . Biol. Chem., 143,575 (1942). (8) Lipton, M.A., a n d Elvehjem, C. A.,Nature, 145,226 (1940). (9) Livshits, hl. I., Proc. Sci. Inst. Vitamin Research U . S. S . R.,3, 184 (1941). (10) Melnick, D., a n d Field, H., Jr., J . Bid. Chem., 127,531 (1939).
