F . M. STRONG AND L. E. CARPENTER, College o f A griculture, U niversity o f W isconsin, M adison, Wis.
T
H E Snell and Strong microbiological assay for riboflavin (10) has been employed successfully by many laboratories on a large variety of biological materials. I t is gener
ally accepted th a t the values so determined compare favor
ably with those obtained by animal and physicochemical methods. However, it has been recognized th a t the micro
biological m ethod gives higher results than other methods on certain types of m aterial, notably cereals and cereal products.
It has been reported (1, 9) th at the starch component of ce
reals, or som e substance associated with it, is responsible for the high values obtained by the microbiological method. According to Scott. R andall, and ‘Hessel (9) the stim ulatory substance is de- stroyed oy digestion w ith taka-diastase, while Andrews, Boyd, and Terry (/) found th at taka-diastase digestion alone did not suffice to avoid the stim ulating effect. Bauem feind, Sotier, and Borult (2) reported th at stim ulatory substances were removed from food materials by extraction with organic solvents and demon
strated th at a number of known fat-soluble compounds are cap
able of stim ulating th e test organism, Lactobacillus caset. An ether-soluble fraction has also been obtained from blood which stimulates the growth of the organism, and a substance is present in alkali-hydrolyzed liver which inhibits it (7).
This paper describes a comparative study of modified pro
cedures proposed for th e riboflavin assay, a study of the ef
fect of pure fa tty acids, glycerides, and other organic mate
rials on th e te st organism, and a simple and effective pro
cedure which has been developed in this laboratory for determining riboflavin in cereal products and other materials.
E x p erim en ta l
The microbiological assays were carried out as origi
nally described (10), except th a t modifications were made in the preparation of the samples for assay.
[A minor alteration in th e preparation of the yeast supplement was also introduced— viz., the first filtration after adding t basic lead acetate was om itted. Instead, the mixture w* s a once made alkaline w ith ammonia, and was then filtered,
procedure is much less laborious. , ___
Entirely satisfactory yeast supplements have also bee p pared from whole autolyzed yeast (Difco Laboratories, Detr ),
C ornstarch« C ontaining 5 M icrofram s per G ram of Added Riboflavin
• C ornstarch itself.contained less th a n 0.20 'ether-so1uble’m aterial from 1 gram of
& Aqueous suspension containing 5 m icrogram s of rib cornstarch. volume made to 100 cc. The mixture was filtered by pouring re
peatedly through a fluted, N o. 40 Whatman filter paper until a clear filtrate resulted. A 50-cc. aliquot of the filtrate was ad
justed to pH 6.6 to 6.8, and diluted to 100 cc. for assay. In the case of a few samples it was found advantageous to filter a second tim e after the pH had been adjusted to 6.6 to 6.8 (cf. T able V III).
Pr o c e d u r e 3, Fi l t r a t io n a n d Et h e r Ex t r a c t i o n. This was the same as procedure 2, except th a t the 50-cc. aliquot of the first filtrate was shaken o u t’ three tim es with 30-cc. portions of ether W ithout removing dissolved ether the aqueous phase was adjusted to pH 6.6 to 6.8, and diluted to 100 cc., and aliquots re-SPThe"ether-soluble fraction of cornstarch was obtained by con
tinuous extraction of a suspension prepared by autoclaving 20 grams of commercial cornstarch for 15 m inutes a t 1 kg. per sq. cm.
(15 pounds per sq. inch) pressure in 200 cc. of 0.1 N hydrochloric acid. Palm itic acid, stearic acid, and tristearin were Eastm an products, and were used as received.
The oleic acid used had been purified by low-temperature crys
tallization from acetone, followed b y distillation in vacuo.
Iodine value, S9.8; calculated, 89.9. Triolein w as prepared from oleic acid purified in this manner, and was subjected to molecular distillation before use. T he linoleic acid was a sam ple which had been fractionated at reduced pressure. T he acid was peroxide-free. Iodine value, 179.5; calculated, 181.1. The oleic acid, linoleic acid, and triolein were a ll preserved b y seaUng in evacuated ampoules im m ediately after distillation. The lecithin tested was a sample of commercial soybean lecithin which had been rendered “oil-free” b y repeated extraction with acetone in a Waring blendor.
R e su lts
The type of difficulty encountered in applying th e original, direct procedure (procedure 1) to a cereal is illustrated in Table I. The drift in the values obtained for whole w heat flour is typical of cereal products in general. The same effect is seen to a greater degree in th e case of corn
starch containing a known am ount of -■ added riboflavin. I t is evident th a t
both stim ulation and inhibition of the bacterial response occur when th e direct procedure is followed. The stim ulating material was evidently n o t removed by th e S cott procedure. However, pro
cedure 3 and th e Andrews procedure resulted in satisfactory assay values.
T he ether-soluble nature of the inter
fering m aterial is shown b y the d a ta in th e last column. Separate assays showed th a t th e stim ulation observed in this case was n o t caused by riboflavin in the ether extract itself.
910 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 14, No. 11
Fi g u r e 1. Ef f e c to f Fa t t y Ac id so n Mic r o b i o l o g i c a l De t e r m i n a t i o n o f Pu r e Ri b o f l a v i n
U p p er. Effect of palm itic acid. R a tio of riboflavin to palm itio acid:
1. 1 to 5200 2. 1 to 1040 3. 1 to 260 4. 1 to 26
Low er. E ffcct of stearic acid. R atio of riboflavin to stea ric acid:
1. 1 to 1000 2. 1 to 400 3. 1 to 200 4. 1 to 50 5. 1 to 5
In view of the above results and of previous work pointing to th e fat-soluble n ature of th e interfering m aterial (2,7), the effect of various known f a tty substances was studied. Fig
ures 1 and 2 summarize th e results obtained with different mixtures of pure riboflavin w ith various fa tty acids. The effect, as indicated by th e deviation of th e recovery values from 100 per cent, was found to depend both on the absolute am ounts of riboflavin and fa tty acid present in th e tube, and on th e ratio between th e two. The bacterial growth was af
fected very m arkedly a t ratios of 1 to 500 or 1 to 1000. A t the 0.05-microgram level of riboflavin this corresponds to only 25 to 50 micrograms of the fa tty acid per tube. In gen
eral, th e widest deviations from 100 per cent recovery were ob
served a t th e lowest level of riboflavin tested. Oleic and stearic acids markedly stim ulated th e bacterial response, while palm itic acid and especially linoleic acid acted as po ten t in
hibitors. These results corroborate, in part, th e work of Bauernfeind et al. (2), who found th a t oleic, stearic, and pal
m itic acids stim ulated the te st organism, while linoleic acid either stim ulated or inhibited, depending on th e am ount used.
T he effect of a m ixture of two fa tty acids was also investi
gated. T he particular m ixture tested, which consisted of oleic and linoleic acids in th e ratio 1 to 0.8, gave very high recoveries when tested a t a level of 3600 p arts of the m ixture to one of riboflavin.
M IC R O G R A M S R IB O F L A V IN P E R T U B E Fi g u r e 2 . Ef f e c t o f Fa t t y Ac i d so n Mi c r o b io l o g i c a l
De t e r m i n a t i o n o f Pu r e Ri b o f l a v i n U pper. Effect of linoleic acid. R atio of riboflavin to linoleic acid:
1. 1 to 4000 2. 1 to 800 3. 1 to 400 4. 1 to 40
Lower. E ffect of oleic acid. R atio of riboflavin to oleic acid:
1. 1 to 4800 2. 1 to 960 3. 1 to 480 4. 1 to 96
T he d ata in Table I I show th a t in contrast to th e free fatty acids neutral fats had little disturbing influence on the micro
biological determ ination of riboflavin. Lecithin gave a mod
era te stim ulation a t high levels (Figure 3).
Although th e p H of th e m edium (6.6 to 6.8) is such th a t the f a tty acids tested should have been present to only a very sm all degree in the form of soaps, it was of interest to try th e effect of substances capable of lowering the surface ten
sion in neutral solution. The synthetic w etting agents listed in Table I I I proved to be inert a t th e concentrations tested. These Aerosols are sodium salts of various alkyl sulfosuccinic acid esters (8,16).
I n order to determ ine w hether the effect of the unsatu- rated acids m ight be attrib u tab le to peroxides or other oxida
tion products, a sample of oleic acid w'as oxidized by bubbling air through it for 15 hours a t room tem perature, and then tested. Substantially th e same results were secured as with highly purified oleic acid. Benzoyl peroxide a»t concentrations of 40 to 800 micrograms per tube also was w ithout effect.
A comparison of th e different procedures as applied to various cereal samples is given in Table IV . I t seems cer
tain th a t the values obtained by th e direct method and by the
November 15, 1942 A N A L Y T I C A L E b l T I O N 911
R iboflavin W ettin g Agent Riboflavin Recovered
Added A dded Aerosol Aerosol Aerosol reasonably concordant results. In most cases procedures 2 and 3 gave essentially the same values.
The recovery of added riboflavin as determined by the results of fluorometric and animal assays on identical samples.
These d ata show definitely th a t the present method is cap according to procedure 2 was 5.0 micrograms per gram (Table IV)-The d ata in Table V III indicate th a t on certain types of material the original direct method yields reliable values, while m any others m ust be handled by the modified proce
dure.
D isc u ssio n
In th e light of the results reported in the present paper and in previous publications, it is probable th a t the “ drift”
frequently encountered in the micro
biological determ ination of riboflavin is caused mainly by free fa tty acids in
The stim ulation and inhibition which operate to produce th e observed drift
flavin gave a greater stim ulation th a n either smaller or larger amounts (Figure 2, lower) is probably to be explained on this basis. The only other reports known to the authors in which oleic acid has been shown to influence bacterial growth are those of Bauemfeind et al. with Lactobacillus casei (2) and of Mueller with the diphtheria bacillus (4). In the latter case it was also found th a t there was an optim um concentration of the acid, and the effective quantities were of the same order of magnitude as those found to be active toward Lactobacillus casei.
Further evidence th a t the drift is attributable to ;a tty acids was secured in carrying out the experiments summarized in hydrolysis (6), cereal samples in the am ounts required for the determination would supply such acids in sufficient quanti
ties to account for the observed effects. Likewise the stim u
latory and inhibitory substances found in blood and in alka
line hydrolyzed liver (7), although n o t y e t identified, could well be oleic acid and linoleic acid, respectively.
From the inform ation now available i t would seem th a t the preparation of cereals and certain other samples for micro Procedure Procedure Procedure Procedure P rocedure Procedure
2 1 2
912 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 14, No. 11
pound. Although th e bacterial method has been shown to m easure th e ribo
flavin contained in a t least some of these combined forms (6), others m ay exist which are n o t so measured. F u rth er
more, riboflavin itself is less likely to be lost in subsequent precipitations, than, for example, riboflavin phos
phate, which has been shown to have alone proved inadequate. E xtraction of th e original m aterial with a fa t sol
mended as being th e m ost generally applicable m ethod, while procedure 2 is regarded as being suitable for m ost materials, notably cereals and cereal products. T he validity of these procedures is supported by (a) lower and more reasonable results on mixed feeds and composited diet samples, which by the direct procedure have often appeared to contain twice as m uch riboflavin, or more, th a n th e sum of th e constituents;
(b) th e absence of drift a t different dosage levels; (c) satis
factory recovery' of added riboflavin; and (d) good agree
m ent w ith th e results of independent assays b y other methods on th e same samples.
The stim ulations and inhibitions studied in this paper tended to be greatest a t th e 0.05-microgram level of riboflavin.
This observation emphasizes th e desirability of calculating th e riboflavin content of unknowns from tubes for which the titra tio n values fall above this level on th e standard curve.
I t has been th e authors’ experience th a t results from tubes containing less th a n 0.05 microgram of riboflavin are prac
tically worthless, and it is the practice in this laboratory to exclude all such values from th e calculation of th e final assay results.
A question raised b y the present paper concerns the accu
racy of riboflavin values previously obtained b y th e original microbiological m ethod. The agreement which has been observed repeatedly between such values and those secured in other ways, together with the direct comparisons shown in Table V III, makes it appear probable th a t on m any samples, especially those of relatively high potency, th e older values are substantially correct. Results previously obtained on cereals, mixed feeds, composited diets, fish meals, and certain other m aterials by th e direct m ethod are very likely too high.
In the future such materials should be assayed b y a suitably modified procedure. The authors are unable a t present to offer any procedure for determ ining riboflavin in blood which' is an im provem ent over th e m ethod previously suggested (18).
The possibility of avoiding interference from fa tty mate
This m ay be attrib u tab le to variations in the physical state—
November 15, 1942 A N A L Y T I C A L E D I T I O N 913 e. g., degree of subdivision—of the suspensions tested. Fi
nally, the fundam entally different solubility characteristics of the fatty acids and th e B-vitamins offer a ready means of separation.
Interference of th e type here described has also been en
countered, and often to an even greater degree (7), in the microbiological determ ination of pantothenic acid {12).
Similar preliminary treatm ent of the samples appears to of
fer a satisfactory solution {15). The nicotinic acid assay {11), on the other hand, is but little disturbed by fatty acids ( W .
S u m m a r y
A study has been m ade of the substances present in cereals and other biological materials which interfere with the determination of riboflavin b y the microbiological method.
I t has been shown th a t the interference is probably due to small am ounts of free fa tty acids, and methods for avoiding their effect have been investigated.
A procedure for the preliminary treatm ent of the sample has been worked out. T he interfering substances are thereby removed, and reliable values for riboflavin are obtained on such materials as cereals and mixed diets which previously have been difficult to assay by the microbiological method.
A ck n o w le d g m e n t
The authors wish to express their appreciation to F. W.
Quackenbush for samples of oleic acid, linoleic acid, triolein, and lecithin; to the American Cyanamid and Chemical
Corporation for samples of th e Aerosols AY, IB, and MA, and to John S. Andrews, Howard J. Cannon, and Miss Jean Collard for carrying out several of the analyses reported in this paper.
L ite r a tu r e C ited
(1) Andrews, J. S., Boyd, H. M., and Torry, D . E., In d. Eng.
Ch e m., An a l. Ed., 14, 271 (1 9 4 2 ).
(2) Bauernfeind, J. C., Sotier, A. L,, and Boruff, C. S., Ibid., 14, 666 (1942).
(3) Caryl, C. R., In d. En o. Chem., 33, 731 (1941).
(4) Cohen, S., Snyder, J. C., and Mueller, J. H., J . B ad., 41, 581 (1941).
(o) Evans, J. W., and Briggs, D . R., Cereal Chem., 18,443 (1941).
(6) Feeney, R. E., and Strong, F. M., J. Biol. Chem., 133, xxxi (1940).
(7) Ibid., 142, 961 (1942).
(8) Kuhn, R., and Rudy, H., Ber., 69, 2557 (1936).
(9) Scott, M. L., Randall, F. E., and Hessel, F. H., J . Biol. Chem., 141,325 (1941).
(10) Snell, E . E ., and Strong, F . M ., In d. En o. Chem., An a l. Ed., 11, 346 (1939).
(11) Snell, E. E., and Wright, L. D., J. Biol. Chem., 139, 675 (1941).
(12) Strong, F. M., Feeney, R. E., and Earle, Ann., Ind. Eno. Chem., An a l. Ed., 13, 5 6 6 (1 9 4 1 ).
(13) Strong, F. M„ Feeney, R. E., Moore, Barbara, and Parsons, H. T., J. Biol. Chem., 137, 363 (1941).
(14) Strong, F. M.t and Krehl, W. A., unpublished work.
(15) Strong, F. M., and Neal, A. L., unpublished work.
(16) Van Antwerpen, F. J., In d. En g. C h e m ., 33,16 (1941).
Pr ese n ted before th e D ivision of Biological C h em istry , J o in t P rogram on Vitam ins, a t th e 103rd M eeting of th e Am erican Ch em ica l So c ie t y, M emphis, Tenn. Published w ith th e approval of th e D irector of th e W is
consin A gricultural E x p erim en t S tatio n . S up p o rted b y a g ra n t from th e Difco L aboratories, D e tro it, M ich.