microbiological assay of vitamin b6 by thin-layer cup

THE JOURNAL OF VITAMINOLOGY 18, 90-96 (1972)

Microbiological Assay of Vitamin B6 by

Thin-Layer Cup-Plate Method with

Saccharomyces carlsbergensis

TAKAKO ITAGAKI AND TOHRU TSUKAHARA1

Laboratory of Nutrition, Niigata Women's College, Niigata (Post No. 950)

(Received March 4, 1972)

The agar-plate diffusion method for determination of vitamin B6 in natural

products was presented, which improved on its weak point of relative insensitivity

by using a thin-layer plate technique.

The optimum conditions for the assay were proposed as follows. Assay

medium: Atkin's basal medium with minor modifications and 1% agar, 0.75mm

plate thickness (thin-layer), pH 5.0 to 6.0. Standard solutions of vitamin B6: pH 5.0

to 6.0. Inoculum size of test organism: 0.075 optical density in final. Incubation:

30•K, 12 to 16hr.

Under our conditions a reproducible, clear and sharply-defined growth zone

on the assay plate was steadily obtained and there was a linearity of the dose

- response to pure vitamin B6 solution over a wide range of 20 to 2,000mƒÊg/ml.

In the statistical analysis of the results the probable errors amounted to only 4

to 5%. The total vitamin B6 values in some natural materials obtained by this

method were as equal as those determined with turbidimetric ones. Also, recovery

experiments were successful enough and the dose-response line for all samples

used hardly had any drift from that for pure vitamin B6 solution.

This method is simple, accurate and adequate for the routine assay of a

large number of samples.

A plate assay technique for the vitamin B6 complex was first developed using Saccharo

- myces carlsbergensis by Jones and Morris (1) and they stated that this method had a precision closely approaching that of the original turbidimetric method. Thereafter, several inf ormations (2-5) on the plate assay method with some modifications have been published. It has not been, however, widely applied to determination of vitamin B6 because of its chief disadvantage of a relative insensitivity to the vitamin.

The present paper describes an agar plate

assay method for vitamin B6 in natural products,

which improves on the weak point of the older

method by using a thin-layer cup-plate technique.

MATERIALS AND METHODS

1. Test Organism

Culture 4228 (ATCC 9080), a strain of

Saccharomyces carlsbergensis, was maintained

on malt extract agar slants. A slant was

cultured for 24 hr at 30•K and was then stored

in the refrigerator for not more than 2 weeks.

1 板垣隆子,塚原叡

90

MICROBIOASSAY OF VITAMIN B6 BY PLATE METHOD 91

2. Media

Medium for Assay-The medium of Atkin

et al. (6) was employed with the following

additions per liter of medium: niacin 2.5mg,

tryptophan 100mg, and agar 10g. The medium

was autoclaved and stored in the ice box until

required.

Inoculum Medium-The liquid basal medium

plus 10 ƒÊg pyridoxine hydrochloride per liter

was used to prepare an inoculum for the assay.

Ten-ml portions of the medium were placed

in test tubes, autoclaved and stored in the

refrigerator.

3. Yeast Inoculum

The organism from the stock culture was

inoculated into the several test tubes containing

10ml of sterile inoculum medium. After incuba

tion at 30•‹ for 16-24hr, the yeast cells were

collected by centrifugation, washed several times

with sterile 0.9% saline, and resuspended in it.

The concentration of the yeast cells was esti

mated with a photometer and was adjusted with

additional saline so as to an optical density of

0.075 in final when inoculated into the assay

medium. This suspension was used for the

seeding of the assay plates.

4. Standard Solution of Vitamin B6

Ten mg of crystalline pyridoxine hydrochlo

ride of special grade were dissolved in 100ml of

25% ethanol to make the pyridoxine concentra

tion exactly 100ƒÊg/ml. This stock culture was

stored in the dark at 5•‹. A standard solution

of pyridoxine might be obtained by an appro

priate dilution of stock solution with a phosphate

buffer solution, pH 5.2, for each experiment.

Fresh standard solutions from pyridoxal and

pyridoxamine were also prepared in a similar

manner.

5. Preparation of Samples for AssayThe assay solutions for total vitamin B6 in

several samples from animal and vegetable sources were prepared by a sulfuric acid extraction technique with minor modifications according to the procedure of Atkin et al. (6). After homogenizing the weighed samples in a Potter

- Elvehjem homogenizer, animal samples were suspended in 90ml of 0.055 N sulfuric acid and vegetable materials in 0.44 N acid. The suspen

sion was autoclaved at 15 lb for 3 hr, then

neutralized to pH 5-6, diluted to 100ml volume,

and finally filtered. This filtrate was used for

the assay.

6. Assay Procedure

The petri dish method was principally used,

because of its simplicity, in the assay design.

The petri dish must be flat-bottomed so as to

obtain an agar plate with uniform thickness;

diameters of 90-100mm are of convenient size.

After melting the assay medium of 45 ml

in a 100-ml tube, the tubes were cooled to

about 48•‹ and maintained at that temperature,

and 5ml of the yeast inoculum was added to

each tube. The contents of each tube were

thoroughly mixed by rotation and stirred with

a pipette to distribute the yeast suspension

evenly throughout the medium. Five ml of the

contents was applied to each petri dish, which

was kept level before plating, and the contents

were allowed to harden to make a thin layer

of the seeded agar. The plates were placed in

a 30•‹ incubator for 2 hr before applying the

test solutions according to the suggestion of

Dennin (2). This drying or preincubation period

improved the definition of the growth zones.

Four stainless steel cups were placed on the

seeded agar surface so that they were at about

60•‹ intervals on a 2.8-cm radius. Four dose

levels of both standard and sample were used.

Ten plates were required for each sample and

they were separated into 2 sets. The sets were

tied together to obtain the dose-response line

for standard and the line for sample. Two

alternate cups on each plate were filled with

the sample dilutions of 2 doses in the ratio of

1: 4, and the remaining 2 cups with 2 standard

dilutions in the same ratio. The sample plates

were replicated 5 times. After incubating the

plates at 30•‹ for 12-16hr, the diameters of the

growth zones were measured by a needle-point

calipers to the nearest 0.1mm. The zone dia

meters for 4 diluents of both sample and stan

dard from 5 cups were each averaged. Then the

theoretical values for the observed data of the

standard were computed by the method of least

squares, and the "line of best fit" was plotted

on a semilogarithmic graph paper. The observ

ed values for the sample were read from the

92 ITAGAKI AND TSUKAHARA

dose-response line to obtain the corresponding

vitamin B6 concentration (mƒÊg/ml) in each dilu

tion of the sample. This concentration was

multiplied by the appropriate dilution factor to

obtain the potency of the original sample. In

this way, the vitamin content of the sample

was calculated for each level of assay solutions

used. The potency of the sample was finally

determined from the average of the values

obtained from dose levels.

RESULTS

1. Establishment of Assay Conditions

Since in a plate assay technique an exact

reading of the diameters of the growth zones is

strongly required, it is fundamentally important

to obtain a reproducible, clear and sharply

defined growth zone. Lees and Tootill (7) have

observed that various physicochemical and biolo

gical factors affect the size, density, and defini

tion of the zones of exhibition on petri dishes.

In the present study these factors were rigor

ously examined and a favourable condition for

vitamin B6 assay by the plate method was

preferred as summarized in Table 1.

Among these factors, pH of assay medium,

depth of seeded agar, and density of seeding of

test organism were three dominant causes that

exerted a great influence on the size, clarity and

sharpness of the zones of exhibition in the vita

min assay. In general, relative times for diffu-

TABLE 1Optimum conditions required for vitamin B6 assay

FIG. 1 Sizes and characters of, growth zones

that develop under the test conditions described

in Table 1

The numerals denote the pyridoxine concentra

-tion. mƒÊg/ml.

sion of vitamin solution before test organism

begins to grow have a effect of inflating the

zone sizes, indicating an enhancement of sensi

tivity of the plate method. Our experience,

however, has shown that in case of vitamin B6

assay by this method any diffusion time is not

necessary for the purpose, because of its relative

high sensitivity to the compound, if only apply

ing a thin-layer technique.

Figure 1 shows the zones of exhibition that

develop under our conditions described above .

They appear as an opaque and double circular

area surrounding the cup; the internal one is

relatively thin in yeast growth; the external one

is so much thick that forms a dike.

2. Precision of the Thin-Layer Plate Me

thod

1) Reproducibility, Dpse-Response Litre

and Assav Scale; of the, Method

The reproducibility of assays by the plate

method was examined under our conditions with

sevetal dose levels of the standard pyridoxine

over a wide range of 20-2,000 mƒÊg/m1. The

experiments were repeated 5 times. As shown

in Table 2, five response values (diameter of

zones of exhibition in mm) for each of the


TABLE 2Reproducibility of thin-layer plate method

standard levels were nearly equal and their

standard deviations were quite small. These

results strongly suggest that the observed values

concentrate near the mean values and are

reliable in reproducibility.

For this method, the existence of a linear

relationship between log dose and zone diameter

is of essential condition. The average response

values for each of the standard levels from 5

cups were plotted against the logarithms of the

concentration of the standard pyridoxine. As

illustrated in Fig. 2, the plotted points were

proportional to the logarithms of the amounts of

pyridoxine placed in the cups. Under the test

conditions, this relationship held with concentra

tions of pyridoxine over a wide range of 20

- 2,000mƒÊg/ml. To ascertain the situation of

these observed points on a straight line of the

dose-response, the theoretical values for the

observed data were computed by the method of

least squares, and the "line of best fit" was also

drawn in Fig. 2. The observed points agreed

well with the computed ones. This fact may

be expressed by the following equation: D=a

log C+ƒÀ, where a and ƒÀ are constants, D is

the zone diameter in mm, and C the vitamin

B6 concentration in mƒÊg/ml.

Similar results were also obtained in the

experiments with both pyridoxal and pyridoxa

- mine as the reference standard. Thus, the stan

dard dose-response line would be applicable to

the assay for any of the three known forms of

vitamin B6. The useful scale in the thin-layer

plate assay should be regarded as ranged from

a concentration of 20 to 2,000mƒÊg of vitamin

B6 per ml, over which the relationship between

FIG. 2 Response of Saccharomyces carlsbergensis

to pyridoxine as measured by the thin-layer plate

method and the "line of best fit" for the plotted

points

D is the zone diameter in mm, C the pyridoxine

concentration in mƒÊg per ml, and 9.0 and -0.6 are

the constant which have been computed from the

observed data by the method of least squares.

log dose and zone diameter has been demon

strated to be linear. Since in the turbidimetric

method the lower limit of sensitivity is 2.5mƒÊg

/ml concentration, that of the thin-layer plate

method may stay in a lowering of one-eighth

the sensitivity.

2) Statistical Errors of the Method

The precision of the plate assay technique

may be demonstrated by the application of

statistical method. Our statistical evaluation of

the assay results showed that when using 5

plates and pure solutions of pyridoxine as the

standard, the probable errors amounted only to

4-5% at highest; for pyridoxal, to 4-8%; and

for pyridoxamine, to 7-12%. These errors in

the conventional plate method for the assay of


other B-group vitamins have been proved to be about 10-15%. The authors, therefore, used to prepare a standard dose-response line for pyridoxine which has the best precision among these compounds. After all, the plate method employing a thin-layer technique was found to be reliable enough in respect of its precision, that is, its reproducibility.

3. Utility of the Thin-Layer Plate Method1) Comparison of Total Vitamin B6

Content of Some Natural Products Assayed by the Plate and Turbidimetric Method

Table 3 gives the results of the estimations made on some natural products from animal and vegetable sources, compared with estimations obtained by the turbidimetric technique. The total vitamin B6 values obtained on these samples agreed well with those determined by

the turbidimetric method, except for a few samples, pork liver and peanut, which were about 10% lower than the latter method. Also, Fig. 3 illustrates an example of practical use of the plate method for the vitamin B6 assay made on rice bran extract. The characters of the growth zones of the sample were quite similar to those of the standard; they were sufficiently clear and sharply-defined.

2) Validity of Assay

To prove the validity of the method, the ability of the assay technique to give theoretical results for pyridoxine added to each sample should be examined. This was tested by adding different amounts of pyridoxine to a sample solution and assaying as previously described. The results are presented in Table 3. The recovery rates of the added vitamin B6 fell

TABLE 3Comparison of vitamin B6 content of some natural products assayed by the plate and turbidimetric method and of results of recovery experiments

All results were reported on the basis of pyridoxine hydrochloride as a reference standard,


FIG. 3 An example of assay of vitamin B6 in

rice bran extract by the thin-layer Plate methad

R1: assay solution of high dose level from rice

bran. R2: the same solution of low dose level in

a quarter the R1 concentration, S1: standard solu

tion of pyridoxine, 800mƒÊg/ml, S2: standard pyri

- doxine diluent, 200mƒÊg/ml.

within 90 to 97%.

The validity of the method was also con

fi rmed by observing the parallelism between the

standard and sample response lines. Any drift

of this line for sample from that for standard

could not be observed, indicating an absence of

any factor interfering the valid assay of vitamin

B6 by the plate method.

DISCUSSION

The use of agar plate method for vitamin

assay was first introduced with a much expecta

tion by Bacharach et al. in 1948 (8) and

thereafter more detailed investigations on the

method have been made by many workers.

The relative insensitivity of the method, however,

has precluded its practical application. In the

plate assay method with S. carlshergensis for

the vitamin B6 complex, its sensitivity to the

compounds is originally successful as compared

with that of the yeast turbidimetric technique.

It seems likely that this considerably high

sensitivitymay be attributed to a rapid diffuseness

of the compounds through the agar plate due to their low molecular weight. Further, the application of a thin-layer technique to the plate method for the assay of vitamin B6 is effective for extending the lower limit of sensitivity of the assay.

The specificity of the yeast microbiological method for vitamin B6 is one of the principal factors affecting the precision of the method. Atkin et al. (6) have already confirmed the specific response of S. carlsbergensis to pyri

- doxine. A little disagreement in the response of the yeast to the three known forms of vitamin B6 was observed in an early study by Fukui et al. (9). On the other hand, Jones and Morris (1) emphasized that the addition of an appropriate amount of tryptophan resulted in uniform response of the yeast to pyridoxine,

pyridoxal, and pyridoxamine, and in the absence of tryptophan the organism response was comparable for the two formers, but was of a different magnitude for the latter. Similar results were also obtained by the present investigation and the addition of tryptophan improved the definition of the enhancement zones. For these reasons, in the plate assay method of vitamin B6 complex the assay medium with tryptophan should always be used.

With a few exceptions the thin-layer plate method gave results of the vitamin B6 content of some foods and other materials which agreed well with those obtained by the turbidimetric method, and added pyridoxine might be recovered from the extracts of natural products without appreciable loss or gain. The estimated values for the various extracts also showed no

perceptible change at different testing levels, i. e., no drift. Although the success of recovery experiments does not establish the validity of an assay method, it is a favourable sign.

These results may support the reliability and specificity of the thin-layer plate method for vitamin B6 determination.

REFERENCES

1. Jones, A., and Morris, S., Analyst, 75, 613 (1950).2. Dennin, L.J., Analytical Microbiology, p. 489,

Academic Press. New York and London, (1963).


3. Kojima, H., Matsuya, Y., Ozawa, H., Konno, M., and Uemura, T., J. Agr. Chem. Soc. Japan, 32, 33 (1958).

4. Takeuchi, S., and Okamoto, S., Vitamins, 32, 522 (1965).

5. Toda, T., and Matsuda, T., Vitamins, 35, 50 (1967).

6. Atkin, L., Schulz, A. S., Williams, W.L., and

Frey, C. N., Ind. Eng. Chem., Anal. Ed., 15, 141 (1943).

7. Lees, K. A., and Tootill, J. P.R., Analyst, 80, 95 (1955).

8. Bacharach, A. L., and Cuthbertson, W. F. J., Analyst, 73, 334 (1948).

9. Fukui, S., Kishibe, T., and Tani, Y., Vitamins, 6, 436 (1953).

microbiological assay of vitamin b6 by thin-layer cup

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