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A REVISED MICROBIOLOGICAL
ASSAY METHOD FOR RIBOFLAVIN
AND PANTOTHENIC ACID -

Thesis for the Degree of M. S.
MICHiGAN STATE COLLEGE

Willard G. McCullough
19-42

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A REVISED MICROBIOLOGICAL
ASSAY'METHOD FOR RIBOFLAVIN
AND PANTOTHENIC ACID

Thesis for Degree of M. S.
Willard G. McCullough
1942

A REVISED MICROBIOLOGICAL
ASSAY METHOD FOR RIBOFLAVIN

AND PANTOTHENIC ACID

A Thesis Submitted to

the
Graduate Faculty

of

Michigan State College
of

Agriculture and Applied Science
as partial fulfillment

for the
Degree of master of Science

Department of Bacteriology and Hygiene

Willard G. McCullough
1942

THESIS

ACKNOWLEDGEUENT:
The writer wishes to express his gratitude to
Dr. W. L. Mallmann and to Dr. C. A. Hoppert for their

advice and guidance.

14388.41

Chemical and physicochemical methods for the quantitative
estimation of riboflavin in biological products involve many
errors.(l) Workers agree that at present biological assay pro-
vides the most reliable method for the estimation of this vit-
amin. Biological assay methods for riboflavin other than the
microbiological method are expensive, time-consuming, and of
limited applicability because of the quantities of the material
required for assay. Further, the value of the Bourquin-Sherman
unit in terms of pure riboflavin as reported in the literature
varies from less than two to approximately five micrograms per
unit.(l,2,3,4,5)

Riboflavin has been shown to be essential for growth of
certain lactic acid bacteria, and the amount of growth has been
shown to be directly prOportional to the concentration of ribo-
flavin in the culture medium.(6) Naturally occurring ribo-
flavin proved more active in promoting growth than did any of
its homologs or isomers. Degradation products produced from
ribbflavin by light were inactive.

Snell and Strong developed an assay method for riboflavin
in natural materials on the basis of the above information.

The bacterium’Lactobacillus helveticus,which is used as
the test organism in a microbiological assay procedure for
riboflavin also requires pantothenic acid.(7,8) A medium ade-
quate for Lactobacillus helveticus except for the absence of
pantothenic acid may be used to assay this vitamin since the

addition of graded amounts of synthetic calcium pantothenate
results in a proportionate increase in lactic acid production.(9)

After an inspection of the media used in the microbiolog-
ical assays for riboflavin and pantothenic acid, the author con-
ceived that a simplification of the assay media would be a con-

tribution in this field since such simplification would lessen

-2-
the time and effort necessary to run assays of these vitamins.
This paper contains a description of the studies made and the
results obtained.

Lactobacillus helveticus was used as the test organism
throughout these studies. To carry stock eultures, the method
used was that of Snell and Strong. Stab cultures of the organ-
ism are carried in yeast-water agar containing one per cent of
glucose. To prepare and carry cultures for assay purposes the
following procedure may be invoked: From the original culture
make a series of stab transfers into yeast-glucose-agar, incu-
bate at 37 Ct for 24 hours, and store in the refrigerator.
Several tubes are reserved as stock cultures, and the others are
used to prepare inoculum for assay. A transfer is made into
lOec. of the basal medium to which is added one microgram of
riboflavin and five-tenths microgram of calcium pantothenate.
After 24 hours incubation at 37°C. this culture is again
transferred into the same medium. This second subculture
is ready for use at the end of a twenty-four hour incube
ation period at 3; C. Preferably this culture should be
used prior to 40 hours incubation. In preparation for
seeding assay tubes, the cells are centrifuged out asept-
ically, resuspended in sterile .85% sodium chloride sol-
ution, and one drop of this cell snapension added to each
assay tube. By this procedure, cells from the agar stab
culture undergo one subculture in liquid medium before
use in routine assay.

EXPERIMENTAL

A series of studies was made to determine the nknimnl

food requirements of Lactobacillus helveticus. Experiment-

ation showed that e tryptic digest of casein with

members of the vitamin B complex added is not

-3-

sufficient to support growth of this organism. However,
the same tryptic digest of casein plus a small amount
of an aqueous concentrate of liver extract and egg a1-
bumen supports growth indifferently well.

Bacto peptone with added dextrose and nutrient agar

supports growth exceedingly well. Bacto tryptone with

the addition of dextrose and nutrient agar supports
grpwth slightly better than peptone. Either of these
media may be used to carry stock cultures if these
cultures are being used frequently. Using yeast-glucose-
agar for stock cultures proves to be better since the
rate of growth is much slower than in either peptone or
tryptone media and allows carrying cultures for a longer
period without necessitating transfer.

Yeast extract plus dextrose supports growth fairly
well. Autclyzed yeast with dextrose added is a better
medium for this organism but appears to be toxic since
initiation of growth occurs slowly.

Addition of asparagine, adenine, and uracil to the
medium used by Snell and Strong for a standard ribofleain
curve stimulated increased acid production.

A medium containing Bacto-peptone with the proteose
fraction removed by precipitation with saturated ammonium
sulfate did not support growth.

After an examination of the amino acid constituents
of an aqueous extract of yeast, whole autolyzed yeast,
and various peptones, an attempt was made to grow lacto-
bacillus helveticus on a medium consisting of amino acids,
vitamins, dextrose, sodium acetate, and inorganic salts.

This medium included the following constituents: inor-

281118 881138- dextrose- sodium 806138138. (”78171118- 88118113931118.

-4-
glycine, alanine, arginine, tryptOphane, riboflavin, panto-
thenic acid, and biotin. No growth resulted. The addition
of guanine, adenine, and uracil to this medium resulted in
growth, but it was slow in appearing.

Further addition of pyridoxine, thiamine hydrochloride,
and para-amino-benzoic acid caused no increased stimulus
of growth.

From the information gathered by the above experi-
mentation, a medium was chosen for an assay procedure.
The following section contains a description of the prep-
aration and use of this medium.
Preparation of Medium:

The riboflavin-free, pantothenic acid-free basal
medium used is a modification of that developed by
others.(lO) It contains photolyzed, sodium.hydroxide-
treated peptone, 0.5%; inorganic salts; cystine, 0.01%;
glucose, 1%; sodium acetate, 0.6%; amino acids; biotin,

500 micromicrograms per 10 cc. medium; nicotinic acid,
0.5 microgram per 19 cc. medium. When riboflavin assays
are made, the medium contains 0.2 micrograms of d-calcium
pantothenate per 10 cc. of medium. When pantothenic acid
assays are made, the medium includes one microgram of
riboflavin per 10 cc. of medium.

Photolyzed Sodium Hydroxide-treated Peptone:

A mixture of 40 grams of Bacto peptone in 250 cc.
of water and 20 grams of sodium in 250 cc. of water is
exposed in a 25 cm. crystallizing dish to light from a
lOO-watt bulb equipped with reflector at a distance of
30 cm. for ten hours, and then is allowed to stand at

room temperature for an additional 14 hours. The basicity

-5-

is neutralized with glacial acetic acid, 7 grams of anhydr-
ous sodium acetate added, and the mixture is diluted to

800 cc. This solution contains the equivalent of 5 per
cent peptone and 6 per cent sodium acetate, which is ten
times the concentration of these materials in the final .
medium. The solution is preserved under toluene in the
refrigerator. The described treatment destroys all of the
riboflavin as demonstrated by the failure of the solution
to show fluorescence when checked in an electrofluorometer.
The above procedure destroys pantothenic acid also.

Inorganic Salts:

Solution 1 consists of 25 grams of dipotassium hydro-
gen phOSphate and 25 grams of potassium dihydrogen phos-
phate dissolved in 250 cc. of water. Solution 2 contains
10 grams of magnesium sulfate heptahydrate, and 0.5 gram of
manganese sulfate tetrahydrate dissolved in 250 cc. of
water‘amd 0.527110%“ EzSoLl-THgo Plus 0.57% 37.14

Cystine:

A solution of cystine hydrochloride containing 1 mg.
of cystine per cc. is prepared and stored under toluene.

Amino Acid Solution:

A stock solution containing 500 micrograms per ml.
of adenine, 500 micrograms per ml. of guanine, and 500 ..
micrograms per ml. of uracil is prepared and stored under
toluene in the refrigerator.

Glycine, alanine, tryptophane, aSparagine, and
arginine are weighed, dissolved in hot water and added
when needed.

Chemically pure anhydrous glucose is added dry as

required.

A ntnnk gnlntinn nnnteininp nicotinic said and hiniix

-6-

is prepared and refrigerated under toluene.

Standard Fiboflavin Solution:

As a stock solution, pure crystalline riboflavin is
weighed and dissolved in warm 0.02 N acetic acid. One
hundred micrograms per cc. is a convenient concentration.
For daily assaying, a more dilute solution is prepared
from the stock solution by dilution with 0.02 N acetic
acid. These solutions are stored under toluene in the
refrigerator and protected from.light. Under such condi-
tions riboflavin is stable. No deterioration could be
detected by periodic examination in an electrofluorometer
over a period of several months. Synthetic d-riboflavin
of at least 99% purity obtained from Merck and Company,
Inc., was uSed as the primary standard for this vitamin
during the entire course of the investigation.

A stock solution of pure d-calcium pantothenate,
obtained from Merck, is prepared in 0.05 M phOSphate
buffer and preserved under toluene in the refrigerator.
Procedure:

The assay fermentations are carried out in ordinary
chemical or bacteriological test tubes containing the
basal medium plus the assay sample in a volume of 10 cc.
A metal rack may be used to segregate the tubes and may
be autoclaved. If 50 assay tubes are to be set up from
stock solutions of the concentrations given above, 50 cc.
of photolyzed, sodium hydroxide-treated peptone solution,
50 cc. of of cystine hydrodhloride solution, 5.0 grams of
glucose, 2.5 cc. of inorganic salt solution number 1, and
2.5 cc. of inorganic salt solution number 2 are mixed.

Sufficient quantity of amino acids and vitamins are added

-7-

to give the following concentrations: glyCine, .02fl;
alanine, .023; tryptOphane, .0075fi; asparagine, .02%;
adenine, 1 mg.%; guanine, 1 mg.%; uracil, l mg.fi; biotin,
500 micromicrograms per 1000. of medium; nicotinic acid,
.5 microgram per 1000. of medium. If a riboflavin assay
is to be made, calcium pantothenate is added to give a
concentration of 0.2 micrograms per 1000. of medium. If
a pantothenic acid determination is desired, riboflavin
is added to give a concentration of one microgram per
lOcc. of medium. The resulting solution is thoroughly
mixed, adjusted to pH 6.8 with sodium hydroxide, and the
mixture diluted to 250cc. Five cubic centimeters are then
pipetted into each of fifty tubes, and a suitable aliquot
of the riboflavin-containing or pantothenic acid-containing
extract added, dependent on which vitamin is being deter-
mined. Lhere necessayy the contents of each tube are
diluted to give a resultant volume of 1000. The tubes
are sterilized by autoclaving at 15 pounds per square
inch for fifteen minutes. Upon cooling the tubes are
ready for seeding. The procedure described in the first
section was used throughout for preparing inoculum and
for seeding tubes with the assay organism.
Standard Curves:

With each set of riboflavin assays duplicate tubes
are set up containing, per 1000. of medium, 0.0, 0.05,
0.075, 0.1, 0.11, 0.15, 0.2, 0.5, and 0.5 microgram of
riboflavin. Growth data obtained from these tubes per-
mits the construction of a standard curve. To construct
such a curve acid production is plotted against ribhflavin

concentration.

-8-

With each set of pantothenic acid determinations
.duplicate tubes are set up containing, per locc. of
medium, 0.0, 0.025, 0.05, 0.075, 0.10, 0.125, and 0.15
micrograms of pure d-calcium pantothenate. A curve is
drawn with calcium pantothenate plotted against acid
production.

Determination of Bacterial Response:

Growth reaponse of this organism to either ribo-
flavin or pantothenic acid may be best measured by titra-
tion of lactic acid produced. Contents of the assay tubes
are transferred to 125cc. Erlenmeyer flasks, loco. of
distilled water added, and the suspension is titrated to
pH 6.8 with 0.1 N sodium hydroxide. Bromothymol blue
serves satisfactorily as an indicator. The end point is
reproducible to :L' 0 . 1cc .

Incubation Period:

All assays herein described were incubated for 48
hours at 57°C. Turbidity of cultures reaches a maximum
within 24 hours, but acid production increases for at
least three days. However, essentially the same results
may be obtained by titrating at the end of 48 hours incubat-
ion as at the termination of a longer incubation period.
Evaluation of Bacterial Response in Terms of Riboflavin:

Response of Lactobacillus helveticus to ribhflavin
in a typical fermentation is illustrated in figure 1.
Within a limited range, acid production is directly
proportional to the concentration of riboflavin; thus,
the stimulus in growth caused by an unknown sample may

'be evaluated in terms of riboflavin by interpolation on

-9-

the standard curve obtained simultaneously with the
growth data on the unknown sample. For the most accurate
results the riboflavin content of assay cultures must
fall on the more linear portion of the standard curve.
Determinations are more valid when samples are assayed

at several levels within the range indicated and the
values obtained averaged.

When pure solutions are being analyzed, it is
possible to determine a few hundredths of a microgram
of riboflavin within a few per cent.

Preparation of Sample for Analysis:

For extraction of riboflavin from natural products,
autoclaving the finely ground material at 15 pounds
pressure per square inch for 15 minutes with a quantity
of water may be used. Extraction with .1 N hydrochloric
acid and subsequent neutralization effects the desired
result. Liquids which are readily soluble in water may
simply be diluted, neutralized if necessary, and assayed
without autoclaving.

A similar procedure is feasible in preparing materials
for pantothenic acid determinations.

Data for a typical analysis of riboflavin is pre-
sented in the accompanying table. values taken from the
literature as given by other investigators are included
as a comparison. The milk assay was made directly.

The yeast was suspended in water and autoclaved at 15
pounds pressure per square inch for 15 minutes. A hydro-
chloric acid extraction of the beef liver was made by
autoclaving, and the insoluble residue was centrifuged

out.

-10..

Data for pantothenic acid assays is not incorporated
here as only a few assays were made. However, the results
obtained indicate good agreement with values recorded in

the literature.

RIBOFLAVIN ASSAY DATA

Riboflavin Found Literature Values

Sample Amount Assazed

 

 

Mgs. per tube per gram micrograms
- microgram, sample per gram
micrograms
Milk 1 50 0.096 1.92
60 0.125 2.05
70 0.155 1.95
Av. 1.96
‘ 1068-200
Milk 2 50 0.079 1.59
60 0.102 1.70
70 0.128 1.85
Av. 1.70
Fresh beef
Liver 2.00 0.078 59.00
5.00 0.150 44.50
4.00 0.169 42.26 40-50
6.00 0.248 41.50
Av. 41.71
Yeast
Extract 0.25 0.058 152
0.50 0.075 144
1.00 0.146 146 145-150
2.00 0.294 147

Av. 147.25

10—

 

 

 

 

cc. .1 N
acid produced
s—o
O . A 1 l 1 J J
0.0 0.1 0.2 0.5 0.4 0.5 0.6

Micrograms Riboflavin per 1000. Medium
Figure 5
Standard curve obtained upon incubation of Lacto-
bacillus helveticus at 57°C. for 48 hours with the
addition of synthetic d-riboflavin to the basal medium
used by Snell and Strong and with the further incorpor-
ation of the following materials: l-asparagine, 0.02%;

adenine, 1 mg. %; and uracil, 1 mg. %.

-11-
, DISCUSSION

That riboflavin and pantothenic acid are essential
for growth of Lactobacillus helveticus is evidenced
by data gathered by a number of independent workers.
Stimulus of growth by the addition of material to the
riboflavin-free or pantothenic acid-free basal medium
may be considered qualitative indication of the presence
of riboflavin or pantothenic acid in that material.
This viewpoint is supported by the structural specificity
required for activity, the identity of flavins isolated
from natural sources, and the inactivity of photolyzed
biological extracts. (11)

It is likely that the bacteria are capable of
utilizing riboflavin in its combined forms. Yeast
and liver contain riboflavin in combined forms; the
bacterial assay yields values comparable to those deter-
mined by other methods.

The presence of unknown growth factors in a sample
might conceivably'cause stimulus of the organism result-
ing in high values for riboflavin or pantothenic acid.
Since the addition of a single pure substance, ribo-
flavin or pantothenic acid, to the basal medium allows
growth through repeated subculture, the medium appears
to contain all of the essential growth factors for
Lactobacillus helveticus. However, the exdstence of
other stimulatory but non-essential substances not in-
cluded in the medium must not be disregarded. In this
work the assumption has been made that the growth effect
of non-essential but stimulatory materials is negligible
in a medium containing subOptimal amounts of an essential

factor such as riboflavin or pantothenic acid.

-12-

Biotin was included in the medium since it has
been shown to be essential for this organism. In fact,
an assay method has been developed using Lactobacillus
helveticus as the test organism for a determingtion
of biotin in biological materials.(l2)

SUMMARY

A description is given for a revised biological
assay method for riboflavin and pantothenic acid which
is based on the essential nature of these substances
for Lactobacillus helveticus. The reliability of the
mehtod is given support by agreement of the assay re-
sults at different levels, specificity of structure
required for activity, and comparison of values obtained
by other methods of biological assay of these vitamins
on the same products.

The assay may be applied to crude extracts or to
the whole ground sample in some cases. Thus, extensive
preliminary purification involving destruction of ribo-
flavin or pantothenic acid is avoided.

This method requires small sample amounts, and no
elaborate equipment. A relatively short period of time
is consumed in such a determination. The accuracy of

the method is of the order of”tten per cent.

10.

11.

12.

BIBLIOGRAPHY

Ellinger, P., ”Lyochromes in the Kidney. With a Note
on the Quantitative Estimation of Lyochromes”,
Biochem. J., 52, 576 (1958)

Bessey, 0.A., "Vitamin G and Synthetic Riboflavin",
J. Nutrition, 15, 11 (1958)

Bourquin, E., and Sherman, H. C., "Quantitative Deter-
mination of Vitamin G (B2)", J. Am. Chem. Soc.,
55, 5501 (1951)

Hawk, Bergeim, and Cole, "Bractical Physiological
Chemistry", 11th ed., p. 902, Philadelphia, P.
Blakiston's Son and Co., 1957

Snell, E. E., Strong, F. E., "A Microbiological Assay
(for Riboflavin", J. Ind. Eng. Chem., Anal. ed., 11, 546
1959 -

Snell, E. E., Strong, F. M., and Peterson, W. H.,
"Growth Factors for Bacteria. Fractionation and
Preperties of an Accessory Factor for Lactic Acid
Bacteria.", Biochem. J., 51, 1789 (1957)

Pennington, D., Snell, E. E., Wielians, R. J., "An
Assay Method for Pantothenic Acid", J. Biol. Chem.,
155, 215 (1940)

Snell, E. E., Strong, F. M., and Peterson, W} E.,
"Pantothenic Acid and Nicotinic Acid as Growth Factors
for L?ctic Acid Bacteria", J. Am. Chem. Soc., 60, 2825

1958

Strong, F. M., Feeney, R. E., and Earle, "A Micro-
biological Assay Method for Pantothenic Acid",
J. Ind. Eng. Chem., Anal. ed., 15, 566 (1941)

Snell, E. E., Strong, F. M., and Peterson, "’. H.,

"Growth Factors for Bacteria. Pantothenic and Nicotinic
Acids as Essential Growth Factors for Lactic and
Propionic Acid Bacteria", J. Bact., 58, 295 (1959)

Snell, E. E., and Strong, F. M., "The Influence of
Riboflavin and Certain Synthetic Flavins on the
Growth of Lactic Acid Bacteria", J. Biochem., 125,
112 (1958)

Shull, G. M., Hutchings, B. L., and Peterson, W. E.,
"A Microbiological Assay for Biotin", J. Biochem.,
142, 915 (1942)

 

MICHIGAN STAT

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