Calcium Pantothenate Assay - Microbiological Turbidimetric Vitamin B12 Assays

Calcium pantothenate assay is a microbial method used in finding the concentration of calcium pantothenate in specific amino acids and vitamins. The article discusses the procedure, results, and discussion of the experiment. The experiment was conducted using Lactobacillus plantarum and vitamin B12 capsules. The concentration of calcium pantothenate in B complex capsules is determined using the curve.

Calcium Pantothenate Assay 1
Microbiological turbidimetric vitamin B12 assays using Lactobacillus plantarum
Section 108 - BI209
Pharmaceutical Microbiology
Instructor: Andrei Bazyleu
Seyeon Jung
300988554
Centennial College
2018.08.06

Calcium Pantothenate Assay 2
Abstract
Calcium pantothenate assay is a microbial method used in finding the concentration of calcium
pantothenate in specific amino acids and vitamins. Calcium pantothenate is a salt from the family
of pantothenate acid that is ideal for human health since it is used in several functions of the
body including metabolism of carbohydrates and fats, cholesterol, steroid hormones alongside
other compounds.
In the laboratory, the quantity of pantothenate in B complex was estimated using vitamin B12
capsules with the quantities ranging between 2 and 10 ng/ml. The pantothenate solution was
already prepared in the laboratory priory carrying out the experiment. Lactobacillus plantarum
was used in the inoculation of the pantothenate-free growth media.
The procedure involved the addition of one drop of organism into all the tubes i.e. analyte, blank
and standard inoculated prior to their incubation at 35⁰C for 24 hours. The known concentrations
(a set of standards) were prepared and a set of unknowns were prepared and their transmittance
calculated by the end of the incubation period using a spectrophotometer with cuvettes. A
transmittance graph is used in the calculation of the concentration of the calcium pantothenate in
the media. The concentration of the calcium pantothenate in B in complex capsules is determined
using the curve.

Calcium Pantothenate Assay 3
Contents
Abstract............................................................................................................................................2
List of Figures..................................................................................................................................4
List of Tables...................................................................................................................................5
Introduction......................................................................................................................................6
Materials and Methods....................................................................................................................7
Pantothenate standard solution....................................................................................................7
Culture used.................................................................................................................................7
Preparation of the medium...........................................................................................................7
Samples used...............................................................................................................................7
Results..............................................................................................................................................8
Discussion......................................................................................................................................10
References......................................................................................................................................11

Calcium Pantothenate Assay 4
List of Figures
Figure 1: Standard curve showing the relationship between turbidimetric measurement (bacterial
growth) and calcium pantothenate concentration-8
Figure 2: Semi-log graph: Log of Concentration against Turbidimetric Measurement-9

Calcium Pantothenate Assay 5
List of Tables
Table 1: Standard curve showing the relationship between turbidimetric measurement (bacterial
growth) and calcium pantothenate concentration-9

Calcium Pantothenate Assay 6
Introduction
Numerous methods used for assay of pantothenic acid been extensively and elaborately been
discussed in the literature. Such methods have most cases been based on the response of the
growth of yeast, bacteria, and chicks to the concentration of vitamins. Studies have been
conducted to discuss the relative benefits and advantages of such methods (Gregory & Hayflick
2017).
One of the previous publications illustrated that pantothenate acid is a requirement growth factor
by Proteus morganii. Proceeding experiments where synthetic crystalline calcium pantothenate
was utilized in a medium that is chemically defined illustrated that the organism showed a
response to an extremely low concentration of the substance; increasing the level of calcium
pantothenate within the limits yielded a corresponding increase in the growth (Wang 2016).
Following such a reaction, the technique of pantothenate has undergone development for its
assay. Due to the fact that the technique has specific desirable characteristics, there is a warranty
in its application to routine assays of pantothenate acid when it comes to natural materials.
Calcium pantothenate assay is applied in the laboratory in establishing the concentration of
calcium pantothenate present in a sample of a vitamin based on the turbidimetric assay. The
media is supplied with calcium pantothenate (Wang et al., 2018).
Lactobacillus plantarum was adopted for inoculation of all the tube in which there were sets
containing 5 tubes each and another four sets composed of 4 tubes each. The appropriate quantity
of the analyte or standard, medium and water were added to every tube. Two of the tubes were
taken as blanks, one is blank uninoculated and the other blank inoculated. The culture is
introduced in all the tubes exclude the blank uninoculated one after which an incubation is done
at a temperature of about 35⁰C for a time period of 24 hours (Kiruthika 2017).

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The transmittance is determined upon the addition of 1 drop of formaldehyde, having the
spectrophotometer at a wavelength of 590 nm both in the standard and analyte cases. The
average is used in the plotting of a standard curve against both the % T and pantothenate
concentration and this is assumed to be the concentration of the calcium pantothenate. This
experiment aims to test the hypothesis that an increase in the concentration of calcium
pantothenate leads to a corresponding increase in the rate of growth.
Materials and Methods
Pantothenate standard solution: Using Difco pantothenate medium which was in the
form of powder, the solution is prepared already at the concentration of 20 ng/ml.
Culture used: Lactobacillus plantarum was adopted and was incubated at 35 degrees Celsius
for a period of 24 hours in Lactobacillus MRS Broth. The substance is centrifuged at 2000 rpm
for at most 10 minutes and the pellet taken and resuspended in a saline of 10 ml before being
vortexed to mix. The solution is then re-centrifuged and the 10 ml saline discarded (Hou et al.,
2018).
Preparation of the medium: 2 sets of 5 tubes are prepared for standard and then marked as
S-1; S-2, S-3… and 2 sets of 4 tubes are prepared for the analyte and marked as A-1, A-2…. This
is followed by the prepared of blank inoculated and then finally blank uninoculated (Wang et al.,
2018). The recommended amount of water, medium, and standard or analyte are added to each of
the tubes.
Samples used: 10 capsules of B complex with vitamin B12 capsules were adopted as samples
that were blended in distilled water which was then diluted to the same concentration as the
dilution standard of the standard solution which was 20 ng/ml.

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Results
The results from the experiment for the turbidimetric measurement were as shown in the table
below:
Tubes Transmittance
Blank (B-U), uninoculated 100%
Blank (B-I), inoculated 100%
S-1 47.8%
S-2 17.5%
S-3 5.7%
S-4 3.6%
S-5 2.4%
A-1 32.8%
A-2 20.6%
A-3 7.0%
A-4 3.6%
Table 1: Relationship between bacterial growth and calcium pantothenate concentration
The relationship between the growth and the concentration of the calcium pantothenate was
determined by generating a standard curve that was used in the assaying material of the assay
medium (Kim, Kim & Park 2016). The values of the correlation were tabulated and plotted as
shown in figure 1.
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
f(x) = − 0.1047 x + 0.4681
R² = 0.752632337796086
Standard Curve
Turbidity
Concentration
Figure 1: Standard curve showing the relationship between turbidimetric measurement (bacterial
growth) and calcium pantothenate concentration

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The results tabulated in table one was used in generating figure one that illustrates the
relationship between turbidimetric measurement and calcium pantothenate concentration. An
increase in the concentration of calcium pantothenate leads to a corresponding increase in the
rate of growth of bacteria as can be observed from the figure 1. The curve correctly lends itself
for the examination of the pantothenic acid content of the extracts derived from the different
materials or substances.
Figure 2: Semi-log graph: Log of Concentration (y-axis) against Turbidimetric Measurement (x-
axis)
The trend line illustrates a very strong correlation for the estimation of the content of extract of
the pantothenate acid from the different materials. It is nevertheless important to determine such

Calcium Pantothenate Assay
10
a curve every time a collection of assays is made as slight derivations have been noticed in the
curve.
Discussion
In this experiment of assay procedure, a test organism is used that has the ability to effectively
respond to the various concentrations of pantothenate relative to the other organisms that have
been used before in other techniques described in the literature (Hedayati, Mahinpoor & Sadri
2018). Growth is noticed when even as low as 0.0004 γ of synthetic calcium pantothenate is
added to each of the tubes of the assay medium.
From such results, it is possible to estimate the content of pantothenate of exceedingly very low
amounts of natural substances, a fact that to some extent may be significant as other non-specific
factors which could have an influence on the test including inhibitory substances and
interference by the opacity or the color can easily be eliminated through dilution (Brandão et al.,
2016).
The procedure of the preparation of the samples for use in the experiment is quite simple with
minimal chances of contamination. The assay medium concentration tends to the chemical
definition and hence sufficiently correlates with accurate duplication.

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11
References
Brandão, A. P., Cooke, R. F., Corrá, F. N., Piccolo, M. B., Gennari, R., Leiva, T., &
Vasconcelos, J. L. M. (2016). Physiologic, health, and production responses of dairy
cows supplemented with an immunomodulatory feed ingredient during the transition
period. Journal of dairy science, 99(7), 5562-5572
Gregory, A., & Hayflick, S. J. (2017). Pantothenate kinase-associated neurodegeneration
Hedayati, H., Mahinpoor, M., & Sadri, M. Studies in the Modification of Dorset-Henley Medium
for the Production of Bovine Tuberculin
Hou, Z., Sun, Q., Hu, Y., Yang, S., Zong, Y., & Zhao, R. (2018). Maternal betaine
administration modulates hepatic type 1 iodothyronine deiodinase (Dio1) expression in
chicken offspring through epigenetic modifications. Comparative Biochemistry and
Physiology Part B: Biochemistry and Molecular Biology, 218, 30-36
Kim, Y., Kim, D., & Park, Y. (2016). Conjugated linoleic acid (CLA) promotes endurance
capacity via peroxisome proliferator-activated receptor δ-mediated mechanism in
mice. The Journal of nutritional biochemistry, 38, 125-133
Kiruthika, R. (2017). Development and Validation of RP-HPLC Method for Rapid Simultaneous
Estimation of Calcium Pantothenate and Biotin in Pure and Tablet Dosage
Form(Doctoral dissertation, Arulmigu Kalasalingam College of Pharmacy, Krishnankoil)
Takahashi, Y., & Mizushima, T. (2017). U.S. Patent Application No. 15/409,155
Wang, C., Liu, Q., Li, H. Q., Wu, X. X., Guo, G., Huo, W. J., ... & Zhang, S. L. (2018). Effects
of rumen‐protected pantothenate supplementation on lactation performance, ruminal
fermentation, nutrient digestion and blood metabolites in dairy cows. Journal of the
Science of Food and Agriculture, 98(6), 2098-2104