A MICROBIOLOGICAL ASSAY METHOD FOR THIAMINE

Transcription

1 A MICROBIOLOGICAL ASSAY METHOD FOR THIAMINE BY CHARLES F. NIVEN, JR., AND KARL L. SMILEY (From the Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca) (Received for publication, May 17, 1943) It is generally recognized that the most useful and convenient means of quantitatively determining members of the vitamin B complex involve microbiological methods. Several methods of this type have been proposed for the assay of thiamine but each one in use today possesses certain inherent disadvantages that limit its value. The fermentation method of Schultz, Atkin, and Frey (1,2), based on the increased rate of alcoholic fermentation by yeast upon addition of thiamine increments, is not adaptable to large scale work. The number of assays that can be carried out is limited by the apparatus. In addition, the pyrimidine cleavage product is active, thus requiring two determinations for each product in order to obtain a true picture of the thiamine content of certain food materials. The method in which Phycomyces blakesleeanus is used as the test organism (3,4) is time-consuming and is subject to error, since the organism is able to utilize the two moieties of the thiamine molecule. Both the pyrimidine and thiazole portions are active in the yeast growth method as proposed by Williams, McMahan, and Eakin (5) ; in addition, this organism is not sensitive to cocarboxylase. The microbiological method with a strain of Staphylococcus aureus as the test organism, as reported by West and Wilson (6), is also subject to error, since this organism is able to synthesize thiamine from the two cleavage products of the vitamin (7). The purpose of this communication is to introduce a thiamine assay procedure which takes advantage of an organism for which neither cleavage product, alone or in combination, is active. As little as 0.01 millimicrogram of thiamine per ml. of medium can be quantitatively detected. Under the conditions tested, cocarboxylase is approximately 40 per cent more active than thiamine, calculated on a molecular basis, thus requiring an enzymatic hydrolysis for precise determinations in some food products. EXPERIMENTAL Organism-The test organism used is a strain of Streptococcus salivarius (Strain S20B). Members of this species are characterized by their ability to synthesize large amounts of a polysaccharide when grown on sucrose or raffinose agar (8,9). These organisms can be serially cultured in a medium containing only chemically defined constituents, consisting of five B vital

2 2 MICRODETERMINATION OF THIAMINE mins and seven amino acids (10). All strains tested required the addition of thiamine to the medium before growth would occur. Stock cultures of the test organism are maintained in the form of stab cultures in a meat infusion agar plus 1 per cent tryptone, 0.1 per cent glucose, 0.2 per cent KEHPOI, 1.5 per cent agar, and excess CaC03. After growth the stab cultures are kept in the refrigerator. Stock cultures should be transferred every 6 weeks. Inoculums for assays are prepared by transferring the culture from the agar stab directly into the basal medium to which 10 millimicrograms of thiamine per 10 ml. have been added. The culture is incubated at 37 for 24 hours, or until good growth occurs, before being used to inoculate the assay tubes. An inoculum for the following day may be prepared by subculturing again in the same medium. Several Basal TABLE I Medium Caseinhydrolysate gm. Thiamine-free yeast extract Glucose Phosphate buffer (0.4 M, ph 7.4) ml. Salt solution* Sodiumthioglycolate mg. Uracil Nicotinic acid y Riboflavin Calcium pantothenate Biotin (methyl ester) Distilled water to ml. * The stock solution of salts consists of 10 gm. of NaCl, 0.8 gm. of MgSOa, 40 mg. of FeS04s7Hz0, and 12 mg. of MnClz in 100 ml. of distilled water. tubes of medium to be used as inoculums may be made up at one time, but care should be taken to steam the medium for several minutes before use in order to insure prompt growth. Basal Medium-The thiamine-free basal medium contains the ingredients listed in Table I. When sufficient thiamine is added to this medium, Streptococcus salivarius grows in intensity and rapidity equal to that in any ordinary laboratory medium. Hydrolyzed Case&-A 10 per cent acid-hydrolyzed, vitamin-free casein solution is prepared in the usual manner with the use of H804 and subsequent removal of the acid with Ba(OH)2. Traces of thiamine, if present, are removed with norit (20 gm. per 100 gm. of casein) at ph 3.0. Since the test organism is able to synthesize tryptophane, it is unnecessary to replenish the hydrolyzed casein with this amino acid.

3 C. F. NIVEK, JR., AND K. L. SMILEY 3 Thiamine-Free Yeast Extract-A 3 per cent thiamine-free yeast extract is prepared by the method of Williams, McMahan, and Eakin (5), with a few modifications. 6 gm. of Difco Bacto-yeast extract are dissolved in 200 ml. of water and autoclaved for 15 minutes. The ph is adjusted to 3.0, 10 gm. of fullers earth are added, and the mixture is shaken for 30 minutes. The fullers earth is then filtered out, and the filtrate adjusted to ph 1.0 and autoclaved another 15 minutes. After cooling, a second 10 gm. portion of fullers earth is added and the mixture is shaken in a mechanical shaker overnight. The fullers earth is again filtered out; 1.5 gm. of KzHPOd are added to the filtrate, which is then adjusted to ph 7.4 and autoclaved again for 15 minutes. The resulting precipitate is filtered out, and the volume adjusted to 200 ml. and sterilized in convenient sized lots. The added phosphate serves the twofold purpose of destroying the remaining traces of thiamine and removing substances which would precipitate out in the complete medium. The test organism will grow satisfactorily in the medium without the thiamine-free yeast extract but when this substance is omitted in the assay procedure there is occasionally an erratic response to increasing quantities of added thiamine and food substances, usually resulting in high assay values. Yeast and tissue extracts freed of thiamine by other means (adsorption with norit; sulfite treatment (2)) have proved to be inferior supplements. Other Medium Constituents-A stock solution of phosphate buffer is made up by mixing equal volumes of 0.4 M KZHPOJ and KHzP04 and adjusting to ph ml. of this buffer solution are used to prepare 100 ml. of medium. Stock solutions of the salt mixture, reducing agent, and vitamins are conveniently made up separately in such concentrations that 1 ml. of each is sufficient per 100 ml. of medium. All stock solutions are sterilized and stored in the refrigerator. Procedure Assays are carried out in standard 6 inch bacteriological test-tubes which have been selected. to be free from scratches and are of constant diameter (16 mm.). These tubes are conveniently supported in a metal rack that may be autoclaved. The simplest procedure is to make up sufficient medium in concentrations double that necessary in the final preparation and to adjust the ph to approximately 7.4 before 5 ml. amounts are placed in each tube. Distilled water is added from a specially cleaned burette in such quantities that each tube will contain a final volume of 10 ml. after the test substance is added. Since thiamine is partially destroyed on heating in alkaline, and even in

4 4 MICRODETERMINATION OF THIAMINE neutral solutions, it is necessary to add the standard vitamin solution, as well as the test substance, aseptically to the medium after autoclaving. A standard thiamine solution is prepared by weighing 10 mg. of thiamine, which has been desiccated to constant weight, into 100 ml. of 0.1 M acetate buffer, ph 4.5. This stock solution may be sterilized by autoclaving 15 minutes and stored in the ice box. The standard solution should be changed at occasional intervals. In order to establish a curve for assay an aliquot of the stock thiamine solution is diluted aseptically in sterile distilled water to a concentration of 1 millimicrogram per ml. Increasing quantities of this diluted solution are added to the sterilized tubes of medium in duplicate so that tubes containing 0, 0.1, 0.2, 0.4, 0.7, 1.0, and 2.0 millimicrograms per tube (10 ml.) are obtained. Samples for assay are added in other tubes in volumes estimated to contain between 0.1 and 0.6 millimicrogram of thiamine. Volumes up to 5 ml. may be used. 0.5 ml. of the inoculum prepared as described previously is mixed with 10 ml. of sterile saline solution. 1 drop of this diluted mixture is used to inoculate each tube of medium. The tubes are then rotated in order to obtain a uniform mixture of thiamine, or test substance, and inoculum throughout the medium. The tubes are incubated at 37 for 24 hours, after which the growth response is measured. Longer periods of incubation do not appreciably alter the assay values. The sensitivity range may be broadened considerably if the glucose is added aseptically along with the thiamine, or test substance, after the basal medium has been autoclaved. This procedure affords a much greater maximum growth, since the initial ph of the medium is maintained at approximately 7.4 during autoclaving. By following this procedure browning of the medium, which might be of hindrance if growth response is to be measured by titrating the developed acid, is avoided. Acetate buffer, either alone or in combination with phosphate, did not prove to be superior to phosphate as a buffer. The need for scrupulously cleaned glassware and materials cannot be overstressed because of the extreme sensitivity of the test organism toward thiamine. It is advisable to use plastic or aluminum caps that can be cleaned well between runs. If cotton plugs are used, they should be covered with a square of cheese-cloth before being inserted into the tubes. The medium should be made up on the day it is used. A standard curve should be established for each day s run. Measurement of Growth Response-Turbidimetric measurements with a photoelectric calorimeter have been found to be more satisfactory for measuring growth response than titrating the developed acid, since the maximum quantity of acid produced is not so large as that which can be attained with

5 C. F. NIVEN, JR., AND K. L. WILEY 5 Lactobacilli. Good results have been obtained, however, by titrating with 0.05 N NaOH, with brom-thymol blue as an indicator. Results A typical curve resulting from response to thiamine is shown in Fig. 1. The thiamine content of the test substance may be determined by compar- I I I I s, I THIAMINE-MILLIMICROGRAMS FIG. 1. Growth response of Streptococcus salivarius to thiamine TABLE Comparative Thiamine Assays of Various Materials Material Dried milk, A... I B... Whole wheat flour... Dried turnip tops.. tomatoes.. Raw skim milk, A.. B... whole milk. Pasteurized whole milk.... II I I - I- Streptococcus salioarius y per grit. or ml Phycomyces blakesbeanus Y Per gm ing the growth response to that produced by thiamine on the standard curve. The average of values obtained at two or more levels is used. Values taken at the extreme lower or upper flat portion of the curve may result in considerable error. A few results from a number of tests on various food products are shown in Table II. The milk samples were prepared by placing 1 ml. of the milk in 100 ml. of 0.1 M acetate buffer, ph 4.5, and autoclaving for 15 minutes.

6 6 MICRODETERMINATION OF THIAMINE The sterile suspension was then diluted aseptically another 10 times in distilled water, from which aliquots were added to assay tubes. The dried milk samples were reconstituted and then treated in a similar manner. The dried vegetable products were prepared by steaming 1 gm. in 70 ml. of 0.1 N H&04 for 30 minutes, followed by neutralizing with sodium acetate to ph 4.5. The material was diluted to 100 ml. and autoclaved for 15 minutes. 1 ml. of the sterile supernatant was diluted to 100 ml. in sterile distilled water from which aliquots were added to the assay tubes. The assay values given agree favorably with those reported in the literature. Also there is a satisfactory agreement in most cases with those found on the same samples with the use of Phycomyces blakesleeanus as the test organism (the latter tests being made by Dr. K. C. Hamner). The results reported mere obtained by averaging the values from two or more levels run in duplicate. In no case was there found any consistent trend, either upward or downward, in the values at different levels. With few exceptions the assay values at different levels agreed within 15 per cent of one another. Occasionally, however, one finds a tube that gives a value entirely out of line with its duplicate, and those found at ot,her levels, which emphasizes the necessity of running more than one level. Recovery Experiments-Extracts of the three dried vegetable products mentioned in Table II were prepared as described above. Pure thiamine was added to parts of the extracts in amounts equal to 5 y per gm. of the original food. The total thiamine content was determined in the controls, as well as in the samples with added thiamine, and the per cent recovery of added vitamin calculated. The results are shown in Table III. The recovery experiments, as shown in Table III, indicate that in some food products there may be certain stimulating substances other than those present in t.he basal medium. For accurate quantitative determinations it may be desirable to add to the medium an extract of the specific food that has been freed of thiamine, eit,her by fullers earth adsorption or by sulfite treatment. Chemical Specificity-Increasing concentrations of 4-methyl-5-/3-hydroxyethylthiazole and 2-methyl-5-ethoxymethyl-6-aminopyrimidine, both alone and in combination, were added to the basal medium, ranging from 1 millimicrogram to 10 y per 10 ml. of medium. The growth which occurred in all tubes was no greater than that found in the basal medium. Experiments with sulfite cleavage (2) with pure thiamine preparations, as well as with food products, consistently showed over 99 per cent destruction of the activity. izdditional tests on the chemical specificity revealed that cocarboxylase is approximately 40 per cent more active than thiamine, calculated on a molecular basis. This has been found to be true on repeated trials with

7 C. F. NIVEN, JR., AND K. L. SMILEY 7 different thiamine preparations. The results cited in Table IV indicate that the increased activity of cocarboxylase cannot be explained on the basis of impurity of the thiamine preparations used. A cocarboxylase solution containing 10 y per ml. was made up in 0.1 M acetate buffer, ph ml. of this preparation were mixed with an equal amount of a 2 per cent taka-diastase solution in acetate buffer. The mixture was incubated at 45 for 6 hours, after which the enzyme was inactivated by steaming for 10 minutes. Solutions of thiamine and cocarboxy- TABLE Recovery Experiments with Dried Vegetables III Material assayed Whole wheat flour Dried tomatoes t,urnip tops Thiamine Cocarboxylase Thiamine added Thiamine found per gm. sample per gm. sample Recovery of added thiamine Y 7 per cent TABLE Comparative Activities of Thiamine and Cocarboxylase after L bij Terent - Treatments Treatment Activity - * 1 per cent taka-diastase, 45, 6 hours. IV Untreated N HCl, IOO, 30 min. Taka-diastase* Untreated 0.1 N HzS04, 121, 20 min. N HCl, loo, 30 min. Taka-diastase* per cent lase of similar concentration were also treated by steaming for 30 minutes in N HCl, and autoclaving at 121 for 20 minutes in 0.1 N H&Sod. The activities of all treated solutions, compared to that of untreated thiamine, are shown in Table IV. Suitable controls were included to correct for the thiamine content of taka-diastase. It can be seen that upon hydrolysis of the thiamine pyrophosphate molecule with the contaminating phosphatase in the enzyme preparation, the activity lowers to approximately the theoretical. Cocarboxylase treated with inactivated taka-diastase showed no decrease in activity.

8 8 MICRODETERMINATION OF THIAMINE Heating cocarboxylase in acid solutions, sufficient to hydrolyze 1 phosphoric acid molecule from the coenzyme (ll), seems to lower the activity only to a slight degree. It is not the purpose of this communication to discuss methods of preparation of samples for thiamine assay. However, from the data cited above it would seem necessary to treat food samples, known to contain large amounts of cocarboxylase, with a phosphatase preparation in order to avoid high results. Taka-diastase digestion has been reported to be desirable for the liberation of most of the B vitamins in food products (12). Taka-diastase digestion of the three dried vegetable products listed in Table III resulted in no significant decrease of the determined thiamine content. DISCUSSION The proposed microbiological assay for thiamine appears to be superior to other procedures now in use. The test organism cannot utilize either or both cleavage products of the vitamin, which might occur in certain processed foods. Furthermore, cocarboxylase is active, but the determined assay levels may be slightly high unless suitable steps are taken for foods having relatively high concentrations of the coenzyme. No explanation is offered for the greater activity of cocarboxylase. One might conclude that cocarboxylase is incorporated directly into the cell of Streptococcus salivarius without previous hydrolysis of the pyrophosphoric ester. Because of the extreme sensitivity of the test organism toward thiamine no interference is encountered due to the turbidity of added food extracts. Milk or milk powder can be assayed with no difficulty when turbidimetric methods are used for measuring growth response. When thiamine is totally excluded, no growth occurs in the basal medium. Attempts to train the test organism to synthesize its thiamine requirements have met with failure. Thiamine supplied in large excess has not been found to be inhibitory. Although only one strain of Streptococcus salivarius was used throughout this study, it is believed that the assay procedure is not dependent upon one particular culture. Twenty different cultures of Streptococcus salivarius have been found to be identical with respect to their growth factor requirements and it is probable that any strain of this species would be equally satisfactory as a test organism in regard to specificity, sensitivity, and accuracy. These organisms can be easily detected and isolated by plat.ing a throat smear on sucrose-gelatin agar as described elsewhere (8). The authors are greatly indebted to Dr. G. W. Lewis of Merck and Company, Inc., for supplies of cocarboxylase, 4-methyl-5-p-hydroxy-

9 C. F. NIVEN, JR., AND K. L. SM.ILEY 9 ethylthiazole, and 2-methyl-5-ethoxymethyl-6-aminopyrimidine, and to Dr. K. C. Hamner, United States Nutrition Laboratory, Cornell Univerversity, for assaying the food materials by the Phycomyces blakesleeanus method. SUMMARY A rapid and specific microbiological assay procedure for the determination of thiamine is proposed, based upon the growth response of Streptococcus salivurius to thiamine. The thiazole and pyrimidine moieties are not active under the conditions tested. Cocarboxylase is 40 per cent more active than thiamine, calculated on a molecular basis. BIBLIOGRAPHY 1. Atkin, L., Schultz, A. S., and Frey, C. N., J. Biol. Chem., 129,471 (1939). 2. Schultz, A. S., Atkin, L., and Frey, C. nt., Ind. and Eng. Chem., Anal. Ed., 14, 35 (1942). 3. Schopfer, W. H., and Jung, A., Corn@. rend. Acad., 204,150O (1937). 4. Bonner, J., and Erickson, J., Am. J. Bot., 26,685 (1938). 5. Williams, R. J., McMahan, J. R., and Eakin, R. E., Univ. Texas Pub., No. 4137, 31 (1941). 6. West, P. M., and Wilson, P. W., Science, 88, 334 (1938). 7. Knight, B. C. J. G., Biochem. J., 31,731,966 (1937). 8. Niven, C. F., Jr., Smiley, K. L., and Sherman, J. M., J. Bact., 41, 479 (1940). 9. Sherman, J. M., Niven, C. F., Jr., and Smiley, K. L., J. Bact., 45,249 (1943). 10. Smiley, K. L., Niven, C. F., Jr., and Sherman, J. M., J. Bact., 45, 445 (1943). 11. Ochoa, S., and Peters, R. A., Biochem. J., 32, 1501 (1938). 12. Cheldelin, V. H., Eppright, M. A., Snell, E. E., and Guirard, B. M., Univ. Texas Pub., No. 4S37, 15 (1942).

10 A MICROBIOLOGICAL ASSAY METHOD FOR THIAMINE Charles F. Niven, Jr. and Karl L. Smiley J. Biol. Chem. 1943, 150:1-9. Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at #ref-list-1